Fungi of The Order Entomophthorales Infecting Aphids in Slovakia

Fungi of the order Entomophthorales
infecting aphids in Slovakia

(Ph.D. thesis)
by
Marek Barta
marekbarta.sav@gmail.com
SLOVAK UNIVERSITY OF AGRICULTURE IN NITRA
Faculty of Agrobiology and Food Department of Plant protection

Resources
Dean of the Faculty: Head of the Department:
prof. Ing. Magdaléna Lacko- prof. Ing. Ľudovít Cagáň, CSc.

Bartošová, CSc.
Fungi of the order Entomophthorales infecting aphids in

Slovakia
(Ph.D. thesis)
Supervisor:
prof. Ing. Ľudovít Cagáň, CSc. Ing. Marek Barta
Nitra, 2004
ACKNOWLEDGEMENT
This work was done under the supervision of prof. Ing. Ľudovít Cagáň, CSc. to whom I
would like to express my deep thank especially for his good advice and guidance throughout
the study, for revising the manuscript and providing helpful suggestions for its improvement,
and finally for his endless enthusiasm encouraging me in my work, and most of all, for the
opportunity to work on the project.
I wish also express my gratitude to prof. Dr. Rudolf Wegnesteiner, BOKU University in
Vienna (Institute for Forest Entomology, Forest Pathology, and Forest Protection) for his
hospitality on the institute, help and guidance in relation to the field sampling in Austria.
PeaDr. Helena Gabčová, Constantine the Philosopher University in Nitra, is thanked for
helping with the Latin diagnosis.
All my colleagues at the Department of Plant Protection deserve also thanks for support in
my work.
I cannot overlook numerous authorities that provided me with valuable reprints. I feel a
need to apologize to the authors of many excellent papers I have no space to cite all of them.
Special thanks belong to my mother and my friends who have always been there to support
me in difficult times.
CONTENTS
CONTENTS
1 INTRODUCTION ………………………………………………………………… 1
2 AIMS OF THE WORK…………………………………………………………….. 3
3 REVIEW OF LITERATURE……………………………………………………….. 4
3.1 FUNGAL DISEASES OF ARTHROPODS………………………………………………….. 4
3.2 ENTOMOPHTHORALEAN INFECTION OF ARTHROPODS……………………………… 4
3.3 CURRENT STATUS IN THE TAXONOMY OF ENTOMOPHTHORALES………………… 5
3.4 APHIDOPHAGOUS ENTOMOPHTHORALES……………………………………………. 8
3.5 BIOLOGY AND ECOLOGY OF ENTOMOPHTHORALES………………………………… 33
3.5.1 LIFE CYCLE OF ENTOMOPHTHORALES……………………………………………... 33
3.5.1.1 Initiation of pathogenesis……………………………………………………………... 35
3.5.1.2 Invasion and colonisation processes during pathogenesis………………………... 37
3.5.1.3 Post-mortem processes of pathogenesis……………………………………………… 38
3.5.1.4 Host’s defence responses to the pathogens…………………………………………... 39
3.5.1.5 Winter survival of Entomophthorales……………………………………………….. 40
3.5.2 EPIZOOTIOLOGY OF ENTOMOPHTHORALES IN APHID POPULATIONS……………......... 42
3.5.2.1 Types of disease prevalence in aphid populations…………………………………... 42
3.5.2.2 Factors governing a disease prevalence and determining a disease character …… 42
3.5.2.2.1 Host populations…………………………………………………......................... 43
3.5.2.2.2 Pathogen populations……………………………………………………............... 45
3.5.2.2.3 Transmission of infection……………………………………………………......... 48
3.5.2.2.4 Environmental factors…………………………………………………….............. 49
3.6 POTENTIAL OF ENTOMOPHTHORALES FOR BIOLOGICAL CONTROL……………….. 52
3.6.1 HISTORICAL……………………………………………………............................ 53
3.6.2 STRATEGIES FOR BIOLOGICAL CONTROL……………………………………........... 54
3.6.2.1 Classical biological control…………………………………………………………… 54
3.6.2.2 Inoculation biological control………………………………………………………… 55
3.6.2.3 Inundation biological control………………………………………………………… 56
3.6.2.4 Conservation biological control……………………………………………………… 59
3.6.3 BIOCHEMICALS AND ENTOMOPHTHORALES……………………………………….. 61
4. MATERIALS AND METHODS……………………………………………………. 62
4.1SURVEY OF ENTOMOPHTHORALES IN APHID POPULATIONS………………………… 62
4.1.1COLLECTING AND HANDLING OF FUNGUS-KILLED APHIDS…………………………….. 64
4.1.2IDENTIFICATION OF ENTOMOPHTHORALEAN PATHOGENS…………………………... 65
4.1.3COLLECTING AND HANDLING OF LIVING APHID SAMPLES……….................……...... 65
4.1.4IDENTIFICATION OF APHID SPECIES……………………………………………....... 66
4.2IN VITRO ISOLATION OF ENTOMOPATHOGENS AND MAINTENANCE OF CULTURES.... 66
4.2.1PREPARATION OF CULTURE MEDIUM………………………………………………. 66
4.2.2ISOLATION OF PATHOGENS……………………………………………………....... 67
4.2.3MAINTENANCE OF CULTURES IN VITRO…………………………………………….. 67
4.3BIOLOGICAL CHARACTERISTICS OF PANDORA NEOAPHIDIS ISOLATES……………... 67
4.3.1SIZE OF PRIMARY CONIDIA……………………………………………………....... 68
4.3.2GERMINATION OF PRIMARY CONIDIA……………………………………………… 68
4.3.3RADIAL GROWTH RATE……………………………………………………............ 68
4.3.4BIOMASS PRODUCTION OF THE ISOLATES………………………………………....... 68
4.3.5VIRULENCE OF THE ISOLATES……………………………………………………... 68
4.3.5.1Obtaining of sufficient amount of infective inoculum………………………………. 68
4.3.5.2Obtaining of disease-free aphids……………………………………………………... 69
CONTENTS
4.3.5.3 Exposing of tested aphids to various quantities of inoculum………………………. 69

4.3.5.4 Establishing of the most favourable conditions to infection during and after the
exposure……………………………………………………………………………….. 70
4.3.5.5 Regularly monitoring of aphid mortality………………………………………......... 70
4.3.5.6 Establishing the dose-mortality relationship………………………………………... 70
4.4 REGULAR MONITORING OF APHID POPULATIONS AND ENTOMOPHTHORALES AT
DOLNÁ MALANTA……………………………………………………………….. 71
4.4.1 CHARACTERISTIC OF LOCALITY…………………..……………………………….. 71
4.4.2 SAMPLING STRATEGY.............................................................................................. 72
4.4.3 DATA ANALYSIS FOR SPATIAL PATTERNS AND SAMPLING PLANS…………………… 74
4.4.4 TRANSMISSION INFECTION EXPERIMENT………………………………………….... 75
5 RESULTS AND DISCUSSION……………………………………………………... 76
5.1 SURVEY OF ENTOMOPHTHORALES IN APHID POPULATIONS………………………… 76
5.1.1 DESCRIPTION OF SPECIES COLLECTED DURING THE SURVEY………………………... 77
5.1.2 DESCRIPTION OF A NEW SPECIES………………………………………………....... 99
5.1.3 A KEY TO APHID PATHOGENIC ENTOMOPHTHORALES……………………………… 104
5.2 BIOLOGICAL CHARACTERISTICS OF PANDORA NEOAPHIDIS ISOLATES.................... 109
5.2.1 SIZE OF PRIMARY CONIDIA....................................................................................... 110
5.2.2 VIRULENCE OF THE ISOLATES................................................................................... 112
5.2.3 GERMINATION OF PRIMARY CONIDIA........................................................................ 114
5.2.4 RADIAL GROWTH..................................................................................................... 117
5.2.5 BIOMASS PRODUCTION OF THE ISOLATES................................................................... 118
5.2.6 VARIABILITY OF PANDORA NEOAPHIDIS ISOLATES..................................................... 119
5.3 APHID-PATHOGENIC ENTOMOPHTHORALES AT DOLNÁ MALANTA......................... 120
5.3.1 PREVALENCE OF APHID-PATHOGENIC ENTOMOPHTHORALES AT DOLNÁ MALANTA...... 120
5.3.2 EPIZOOTIC PATTERNS OF ENTOMOPHTHORALEAN DISEASES AND SPATIAL
DISTRIBUTION OF APHIDS ……………………………………………………….……… 129
5.3.3 NON-CROP VEGETATION AS RESERVOIR FOR THE PATHOGENS IN LANDSCAPE WITH
SPECIAL ACCENT TO NETTLE PATCHES…………………………............................... 143
6 CONCLUSIONS…………………………………………………………………... 149
7 CONTRIBUTION OF THE WORK FOR PRACTICAL USE……………….………….. 151
8 SUMMARY………………………………………………………………………. 152
9 SÚHRN................................................................................................................... 154
10 REFERENCES CITED…………………………………………………………….. 156
11 APPENDIX………………………………………………………………………. 189
OBSAH
OBSAH
1 ÚVOD …………………………………………………………………………… 1
2 CIELE PRÁCE……..........……………………………………………………….. 3
3 LITERÁRNY PREHĽAD………….....…………………………………………….. 4
3.1 HUBOVÉ OCHORENIA ČLÁNKONOŽCOV………………………..…………………….. 4
3.2 PATOGÉNY Z RADU ENTOMOPHTHORALES NA ČLÁNKONOŽCOCH…….…………… 4
3.3 SÚČASTNÝ STAV V TAXONÓMII HÚB Z RADU ENTOMOPHTHORALES……………… 5
3.4 PATOGÉNY Z RADU ENTOMOPHTHORALES NA VOŠKÁCH………...…………………. 8
3.5 BIOLÓGIA A EKOLÓGIA HÚB Z RADU ENTOMOPHTHORALES…….………………… 33
3.5.1 ŽIVOTNÝ CYKLUS HÚB Z RADU ENTOMOPHTHORALES……………………………... 33
3.5.1.1 Iniciácia patogenézy………........……………………………………………………... 35
3.5.1.2 Invázia a kolonizácia hostiteľa počas patogenézy…………...........……………... 37
3.5.1.3 Priebeh patogenézy po smrti hostiteľa…………….………………………………… 38
3.5.1.4 Obranné mechanizmy hostiteľa……….................…………………………………... 39
3.5.1.5 Prezimovanie húb z radu Entomophthorales…………………….......…………….. 40
3.5.2 EPIZOOTIOLÓGIA ENTOMOPHTHORALES V POPULÁCIÁCH VOŠIEK…………..…......... 42
3.5.2.1 Základné typy hubových ochorení v populáciách vošiek…………………………... 42
3.5.2.2 Faktory riadiace prevalenciu a priebeh hubových ochorení ….............................… 42
3.5.2.2.1 Populácie hostiteľov………...……………………………………......................... 43
3.5.2.2.2 Populácie patogénov……….……………………………………………............... 45
3.5.2.2.3 Prenos infekcie v populáciách………….....…………………………………......... 48
3.5.2.2.4 Faktory prostredia….........…………....………………………………….............. 49
3.6 PERSPEKTÍVA ENTOMOPHTHORALES V BIOLOGICKEJ OCHRANE……....………….. 52
3.6.1 Z HISTÓRIE……..………………………………………………............................ 53
3.6.2 STRATÉGIE BIOLOGICKEJ OCHRANY………….......…………………………........... 54
3.6.2.1 Klasická biologická ochrana…..……………………………………………………… 54
3.6.2.2 Inokulačná biologická ochrana…………….………………………………………… 55
3.6.2.3 Inundačná biologická ochrana……..………………………………………………… 56
3.6.2.4 Konzervačná biologická ochrana………..…………………………………………… 59
3.6.3 BIOCHEMIKÁLIE A ENTOMOPHTHORALES……………………………………….. 61
4. MATERIÁLY A METÓDY……….......……………………………………………. 62
4.1 PRIESKUM ENTOMOPHTHORALES V KOLÓNIÁCH VOŠIEK………...………………… 62
4.1.1 ZBER A MANIPULÁCIA S PARAZITOVANÝMI VOŠKAMI……….......…………………….. 64
4.1.2 IDENTIFIKÁCIA HÚB Z RADU ENTOMOPHTHORALES……….........…………………... 65
4.1.3 ZBER ŽIVÝCH VOŠIEK A MANIPULÁCIA S NIMI …................…….................……...... 65
4.1.4 IDENTIFIKÁCIE DRUHOV VOŠIEK………....……………………………………....... 66
4.2 IN VITRO IZOLÁCIA ENTOMOPATOGÉNOV A UCHOVÁVANIE KULTÚR......................... 66
4.2.1 PRÍPRAVA KULTIVAČNÝCH MÉDIÍ……....…………………………………………. 66
4.2.2 IZOLÁCIA PATOGÉNOV………………………......……………………………....... 67
4.2.3 UCHOVÁVANIE KULTÚR IN VITRO…………........………………………………….. 67
4.3 BIOLOGICKÉ VLASTNOSTI IZOLÁTOV DRUHU PANDORA NEOAPHIDIS …….………... 67
4.3.1 VEĽKOSŤ PRIMÁRNYCH KONÍDIÍ………………………………………………....... 68
4.3.2 KLÍČENIE PRIMÁRNYCH KONÍDIÍ…………….......………………………………… 68
4.3.3 RADIÁLNY RAST IZOLÁTOV…………………......…………………………............ 68
4.3.4 PRODUKCIA BIOMASY IZOLÁTOV……………...........…………………………....... 68
4.3.5 VIRULENCIA IZOLÁTOV……….........……………………………………………... 68
4.3.5.1 Produkcia infekčného inokula……………......................................…………………. 68
4.3.5.2 Chov vošiek…………........…………………................................................................. 69
OBSAH
4.3.5.3Expozícia vošiek rôznym koncentráciám inokula………................………………. 69

4.3.5.4Zabezpečenie optimálnych inkubačných podmienok po inokulácii……....……….. 70
4.3.5.5Pravidelné sledovanie mortality testovaných vošiek…………..........…………......... 70
4.3.5.6Odhad letálnej koncentrácie LC50…………….................…………………………... 70
4.4SLEDOVANIE POPULÁCIÍ VOŠIEK A ENTOMOPATOGÉNOV NA LOKALITE DOLNÁ
MALANTA……………...........………………………………………………….. 71
4.4.1 CHARAKTERISTIKA LOKALITY…………………..…………..…………………….. 71
4.4.2 METÓDA HODNOTENIA POPULÁCIÍ............................................................................ 72
4.4.3 ANALÝZA PRIESTOROVÉHO ROZMIESTNENIA VOŠIEK A METÓDY HODNOTENIA
POPULÁCIÍ…………...............................................................................…………
74
4.4.4 EXPERIMENTÁLNY PRENOS INFEKCIE MEDZI VOŠKAMI………...………………….... 75
5 VÝSLEDKY A DISKUSIA………………………......……………………………... 76
5.1 PRIESKUM ENTOMOPHTHORALES V KOLÓNIÁCH VOŠIEK …..……………………… 76
5.1.1 POPIS A CHARAKTERISTIKA DRUHOV NÁJDENÝCH POČAS PRIESKUMU………..……... 77
5.1.2 POPIS NOVÉHO DRUHU PLESNE Z RADU ENTOMOPHTHORALES………….………....... 99
5.1.3 KĽÚČ NA URČOVANIE PATOGÉNOV VOŠIEK Z RADU ENTOMOPHTHORALES……..…… 104
5.2 BIOLOGICKÉ VLASTNOSTI IZOLÁTOV DRUHU PANDORA NEOAPHIDIS...................... 109
5.2.1 VEĽKOSŤ PRIMÁRNYCH KONÍDIÍ............................................................................... 110
5.2.2 VIRULENCIA IZOLÁTOV............................................................................................ 112
5.2.3 KLÍČENIE PRIMÁRNYCH KONÍDIÍ............................................................................... 114
5.2.4 RADIÁLNY RAST IZOLÁTOV...................................................................................... 117
5.2.5 PRODUKCIA BIOMASY IZOLÁTOV.............................................................................. 118
5.2.6 VARIABILITY IZOLÁTOV DRUHU PANDORA NEOAPHIDIS ............................................. 119
5.3 ENTOMOPHTHORALES V KOLÓNIÁCH VOŠIEK NA LOKALITE DOLNÁ MALANTA..... 120
5.3.1 PREVALENCIA ENTOMOPHTHORALES V KOLÓNIÁCH VOŠIEK.......................................... 120
5.3.2 CHARAKTER EPIZOÓTIÍ V KOLÓNIÁCH VOŠIEK A PRIESTOROVÉ ROZMIESTNENIE
VOŠIEK ……………………………………………………….............................……… 129
5.3.3 NEPLODINOVÁ VEGETÁCIA AKO REZERVOÁR PATOGÉNOV V AGROEKOSYSTÉME
S DÔRAZOM NA PORASTY PŔHĽAVY………………………….................................. 143
6 ZÁVERY………..........………………………………………………………….. 149
7 PRÍNOS POZNATKOV Z PRÁCE PRE PRAX……………….…...................………. 151
8 ANGLICKÝ SÚHRN……...………………………………………………………. 152
9 SLOVENSKÝ SÚHRN.............................................................................................. 154
10 ZOZNAM POUŽITEJ LITERATÚRY…….......…………………………………….. 156
11 PRÍLOHY………………………..………………………………………………. 189
1. INTRODUCTION
1. INTRODUCTION
A co-evolution of various communities of organisms in a common space led naturally to
manifold interactions being developed among particular members of the communities.
Trophic interactions as elementary links connect single organisms within the populations, thus
producing a complex structure of the food web. In reality, all terrestrial communities based on
living plants are composed of at least three or four interacting trophic levels; the primary
producer level (plant), the primary consumer level (herbivorous pest), the secondary
consumer level (natural enemies of the herbivore), and the tertiary consumer level
(hyperparasites). In natural ecosystems the herbivores are usually present in their habitats in
low numbers, since their natural enemies keep them under control. Generally, the
interrelationships among plants, herbivores, and enemies of herbivores are characterised by
the food web stability. This is common in relatively stable or permanent habitats like forests,
orchards, and pastures, where stable interrelationships can develop between “ever-present”
herbivores and the natural enemies. Conversely, in temporary man-managed habitats like
agricultural crops, due to low habitat stability, the effectiveness of natural enemies is
enfeebled and they rarely play a significant role in the population regulation of their host. The
continuously changing pattern of host plants and herbivore complex makes survival of the
natural enemies very tenuous. While herbivore populations in such habitats tend to increase
rapidly in order to be able to utilize host food plants that are easily available, the natural
enemies usually do not have time to become established in the host population before it or its
habitat is gone. As a consequence of the pest outbreaks serious damages of crops are recorded
annually. Therefore, a routine control of the organisms performed within a complex of
strategies and practices of Integrated Pest Management (IPM) is an inseparable part of
agricultural production.
Aphids as plant feeders are a significant component of entomofauna in agroecosystems.
Aphids belong to the insect family Aphididae, within the order Homoptera. In the world there
are nearly 4000 aphid species, mostly in the temperate regions, with about 800 species living
in Slovakia /PAŠEK 1954, DIXON 1998/. As individuals they are small and inconspicuous.
However, they frequently become so numerous that the number of feeding on the leaves and
shoots on 0.4 hectares of ground is 2000 million /DIXON 1973/. Colonies of aphids cause crop
losses by extracting of phloem sap mostly from leaves and stems of host plants, and excluding
honeydew. In addition, aphids are one of the most active vectors of various plant viruses.
Their feeding can result in deformation, wilting, even in necrosis of host tissues depending
upon an infestation level. Infested plants are often coated with shiny honeydew secreted by
aphids, and whitish cast aphids’ skins. The honeydew and the skins on plants give a good
background for flourishing of numerous saprobes. Aphids, thank to their high variability and
specificities of their life cycle, may create forms resistant against frequently applied pesticide
groups. The development of pesticide resistance in aphid populations highlights the
importance of biological control as a pest management tactic. Moreover, it is generally
recognised that some chemical pesticides contaminate groundwater and enter the food chains
that have an impact on a wide range of organisms. Furthermore, pesticides can pose hazards
to the animal health and to the users spraying the chemicals. Keeping that in the mind many
farmers and food producers have been interested in biological strategies of pest management
with the intention to produce residue-free provisions and to eliminate pesticide detrimental
effects on the human health. The biological strategies of IPM reckon on the utilisation of
various groups of natural enemies. In nature aphids often serve as a prey for predators from
orders: Coleoptera, Diptera, Hymenoptera, Nueroptera, and Heteroptera; parasitoids from
order Hymenoptera; or as a substrate for parasiting fungi from the classes Zygomycetes (order
Entomophthorales) and Hyphomycetes /Daniel and Sullivan 1988, Frazer 1988, Starý 1988a,
1988b/. A short generation time (an infection cycle) of fungal entomopathogens and their
1
1. INTRODUCTION
ability to rapidly generate numbers of infection units that are far greater than needed to insure
high levels of host infection make them superior to parasitic and predatory insects, which
have much lower intrinsic rates of reproduction. Entomopathogens, generally, are capable of
infecting several of their hosts’ life stages, unlike many parasitoids and predators, which
select their prey on the basis of size or developmental stage. On the other hand, fungal
pathogens and insect parasitoids may compete within the host tissue (so called “apparent
competition”). The pathogens usually out-compete younger parasitoids, while elder
parasitoids are often capable of completing their development within fungus-infected insects.
Interest in using the insect pathogens as control agents within IPM programs has generated a
research in quest of development of fungal pathogens as microbial insecticides. Much of this
research has been focused on the selection, isolation, development, and production of highly
pathogenic species/strains with genetic characteristics favouring their storage and usage. This
orientation has allowed the pathogens to be applied to target hosts as replacements for
insecticides. Although some successes were noted, the majority of attempts to use the fungal
pathogens as a substitute for insecticides resulted in inconsistent responses. Mostly, the
pathogens were treated as direct substitutes for chemical insecticides and were applied
without an adequate understanding of their population biology or their interactions within a
local environment. Consequently, this lack of understanding caused the field of epizootiology
to be a major research priority in the insect pathology. Nowadays the fungal biological control
is an exciting and rapidly developing research area with implications for plant productivity,
food production, as well as animal and human health. This area has an interdisciplinary
character and includes ecology, genetics, pathology, physiology, mass production,
formulation, and application strategies. Recently, in the light of new knowledge of
host/pathogen biology and the environmental factors affecting the biology, various strategies
of fungal exploitation have been developed and discussed, including classical, conservation,
inoculation, and inundation biological control.
Out of the group of fungal pathogens of aphids the Entomophthorales are apt to be
employed as perspective biocontrol agents of aphids. It is especially because of their short
infection cycle, high reproductive rate, as well as obvious and dramatic epizootics that may
occur almost overnight. One of particularities of Entomophthorales, which allow them a fast
spreading, is an active projecting of conidia from conidiophores. Entomophthorales have
another characteristics that make them valuable mortality agents of aphids. Most of the
species are closely host specific so they pose no significant threat to non-target insects. Some
of them are capable of being cultured or produced in the laboratory or mass-scale. A
technology of mass production is managed for some species, and certain advances have being
made recently in others. The fungi have been studied intensively in laboratories and tested in
a small-scale in the field. However, there are some shortcomings of Entomophthorales as
biocontrol agents coming from a fragility of infective conidia, a dormancy and an
asynchronous germination of resting spores, as well as a complexity of infection cycle
requiring successively: a contact between an infective spore and a host cuticle, a germination,
a penetration of germ tube through a cuticle and, finally, a sporulation. Moreover, all these
phases are highly dependent upon external conditions.
In Slovakia, aphids belong unquestionably to important group of pest insects causing a
direct and indirect damage in agriculture. A control of aphid populations is focussed
exclusively on application of chemical pesticides, though predators and hymenopteran
parasitoids have closely been studied with an aim for biological control. Unfortunately, the
Entomophthorales have been overlooked despite their great potential as control agents. The
aim of this study was predominantly to make the first steps to the knowledge of the fungi in
the country, involving a species spectrum, a general biology and ecology, and a potential as
natural control agents in agricultural ecosystems.
2
2. AIMS OF THE WORK
2. AIMS OF THE WORK
 to collect information about entomophthoralean fungi associated with aphid populations in

the world
 to carried out a survey of aphid colonies in natural ecosystems and agroecosystems to

determine a species spectrum of entomophthoralean fungi under conditions of Slovakia
 to compare the pathogen diversity in Slovakia with that in the world
 to obtain in vitro isolates of some aphid-pathogenic fungal species
 to examine some phenotypic features of P. neoaphidis isolates, including a primary

conidium size, a germination of primary conidia, a virulence, a biomass production, and a
radial growth, in order to evaluate an intraspecific variability
 to evaluate an occurrence of entomophthoralean fungi in selected aphid populations on

crop and non-crop vegetation on the same locality
 to examine epizootic patterns of the most important fungi active in the aphid populations
 to evaluate the spatial distribution of aphids and fungal diseases within a host plant habitat
to determine spatial patterns and to develop optimal sample size curves for aphid cadavers
of selected aphid species
 to evaluate a possible role of non-crop vegetation as a reservoir of aphid-pathogenic

Entomophthorales in agricultural landscape and to study a possible transmission of fungal
infection between various aphid species
3
3. REVIEW OF LITERATURE: 3.1 Fungal diseases of arthropods
3. REVIEW OF LITERATURE
3.1 FUNGAL DISEASES OF ARTHROPODS

Fungal diseases in insect populations are common and widespread. They often decimate
insect populations in spectacular epizootics and thus attract man’s attention. Many of these
fungi are considered an important factor regulating pest insect populations /CARRUTHERS and
SOPER 1987/. Insect pathologists have been interested in the fungal pathogens of their
respective host groups for more than 100 years. Until know at least 90 genera and more than
700 species of entomopathogenic fungi have been identified as closely associated with
invertebrates, principally insects, but only 10 of these have been or are currently being
developed for insect control /e.g. ROBERTS and HUMBER 1981, HAJEK and ST. LEGER 1994,
WRAIGHT et al. 2001, BATEMAN and CHAPPLE 2001/. Within the wide Kingdom of fungi,
virtually every major fungal taxonomic group, except higher Basidiomycetes and
dematiaceous Hyphomycetes, has members pathogenic to insects /ROBERTS and HUMBER
1981/. Entomopathogenic species are distributed predominantly among classes of
Chytridiomycetes, Oomycetes, Trichomycetes, Zygomycetes, and Dueteromycetes
(Hyphomycetes) /PAPIEROK and BREY 1990/. As a matter of fact most species attacking
terrestrial insects belong to the class Hyphomycetes and the order Entomophthorales
(Zygomycetes), while those attacking aquatic insects are generally from the Chytridiomycetes
and Oomycetes /BUTT and GOETTEL 2000/. Although these microbial pathogens share much
in common with viruses, bacteria, and microsporidia, there are many excellent differences and
the most fundamental is the route of infection. Fungal entomopathogens almost always attack
their hosts by direct penetrating the exosceleton and do not require ingestion with subsequent
infection through the gut wall. In contrast, viral, bacterial, and microsporidian pathogens of
insects or other arthropods infect their host primarily via the digestive tract. They must be
ingested and infect the host through the gut wall. Most bacterial entomopathogens infect
through the gut, although bacteria could also gain access to the haemocoele through cuticular
wounds. The difference in the mechanisms of host infection implies that hosts with sucking
mouth parts (orders of Homoptera and Heteroptera) are not attacked by bacteria, viruses or
microsporidia and fungi are only significant microbial pathogens of the hosts /CARRUTHERS
and SOPER 1987, HUMBER 1990/.
Aphids can be attacked by entomopathogens from two different classes. They are
Zygomycetes and Hyphomycetes, however the entomophthoralean fungi from the class
Zygomycetes are the major fungal pathogens of aphids /HUMBER 1990/. Entomopathogenic
Hyphomycetes includes hundreds of species, but just few of them are specific for aphids. For
instance, Verticillium lecanii (Zimmermann) Viégas is one of the most important aphid
pathogenic Hyphomycetes. V. lecanii can attack a broad spectrum of insects in both tropical
and temperate regions, but it is distributed especially in tropical regions /HALL 1984, HUMBER
1990/. It rarely affects aphids under field conditions /e.g. FENG et al. 1990, HUMBER 1990,
HATTING et al. 1999a, ABDEL-MALLEK 2003/. Besides V. lecanii, also Beauveria bassiana
(Balsamo) Vuillemin can seldom infect aphids in natural sites /e.g. FENG et al. 1990, HUMBER
1990, HATTING et al. 1999a, ABDEL-MALLEK 2003/. Both the species, V. lecanii and B.
bassiana, have been successfully commercialised and registered for the use in greenhouses
against whiteflies, aphids, and thrips /WRAIGHT et al. 2001/.
3.2 ENTOMOPHTHORALEAN INFECTION OF ARTHROPODS

The class Zygomycetes comprises two orders, Mucorales and Entomophthorales. Fungi
within the first order are typical saprophytes and are less often or rarely parasitic. The
entomophthoralean fungi are spread all over the world in terrestrial habitats except members
4
3. REVIEW OF LITERATURE: 3.2 Entomophthoralean infection of arthropods
of the genus Ancylistes occurring in waters /BAŁAZY 1993/. At present the order
Entomophthorales consists of 5 families (except Basidiobolaceae) with a total of 20 genera
and 200-300 species /BAŁAZY 1993, KELLER 1987a, 1991, 1997, EILENBERG 2002/. Fungi of
the order have been identified from many insect orders, including Diptera, Homoptera,
Lepidoptera, Coleoptera, Heteroptera, Hymenoptera, Orthoptera, or Dermaptera /BAŁAZY
1993, KELLER 1987a, 1991/. However, the fungi are also known from mites (Acarina)
/KELLER 1997/ and recently from Collembola (Apterygota) /KELLER and STEENBERG 1997,
STEENBERG et al. 1996/.
Fungi from the order Entomophthorales possess several noteworthy characteristics
favouring their use in the biological control. In comparison with Hyphomycetes,
Entomophthorales are able to establish epizootics quickly due to their great capacity for
multiplication and expansion in host populations /LATGÉ et al. 1983, EILENBERG 2002/. Their
conidia are forcibly discharged from conidiophores, which develop on the host surface from
hyphal bodies by penetrating and elongating through the cuticle. The entomophthoralean
fungi may multiply as protoplasts and/or hyphal bodies after having invaded a host and the
hyphal bodies can develop into thick-walled spores (resting spores) inside a cadaver. These
resting spores allow the fungus to survive adverse conditions and persist in the environment
/PAPIEROK and HAJEK 1997/. The Entomophthorales are typical of a high specificity towards
particular host species and generally of low negative impact towards non-target organisms
/LATGÉ and PAPIEROK 1988/. Close associations with foliar insect or mite hosts is further
characteristic of the group of fungi /EVANS 1989/. Entomophthoralean conidia are of larger
size with mucus layer on their surface and they sporulate and germinate quickly. Relatively
small number of conidia are produced per cadaver, but fewer conidia are required for
infection initiation /PELL et al. 2001/. Conversely, hyphomycete fungi, in general, tend to have
wide host ranges and epizootics usually occur only in insect populations in soil /KELLER and
ZIMMERMANN 1989/. Conidia are smaller and produced in higher quantities per cadaver, not
actively discharged, usually without mucus on surface, and many conidia are required for
infection /PELL et al. 2001/.
Entomophthorales are considered a major pathogenic group of sap sucking aphids.
Bacterial and protozoan infections have not been demonstrated in aphids /LATGÉ and
PAPIEROK 1988, HUMBER 1990/, but recently some viruses have been found, which decrease
the longevity of the infected aphid individuals /D’ARCY et al. 1981/. However, no viral
epizootics in aphid populations have ever been reported /LATGÉ and PAPIEROK 1988/.
3.3 CURRENT STATUS IN THE TAXONOMY OF ENTOMOPHTHORALES

Based on the presence of zygospores and a coenocytic mycelium, which, however, may
become divided by septae into segments and often be fragmented into hyphal bodies, the
order Entomophthorales is placed in the phylum Zygomycota and the class Zygomycetes.
Within Zygomycetes, the order Entomophthorales is characterised by the repetitive discharge
of conidia and the ability to discharge conidia actively /WATERHOUSE 1973/.
The classification of the families and genera within Entomophthorales has been disputed
recently. The order Entomophthorales is, in all probability, monophyletic /JENSEN et al. 1998/
and at present the following 5 families are recognised within the order (Figure 1):
Completoriaceae (Humber), Meristacraceae (Humber), Ancylistaceae (v. Thieghem),
Entomophthoraceae (Nowakowski) and Neozygitaceae (Ben-Ze’ev et Kenneth) /EILENBERG
2002/. Only the latter 3 families contain arthropod-pathogenic species /HUMBER 1989,
KELLER 1999/. In this work also we consider the family Basidiobolaceae (Engler et Gilg) a
part of the order Entomophthorales. However, recent phylogenetic studies show that the genus
probably does not belong to Entomophthorales, despite the actively discharged conidia, and
confirm that the genus is more related to chytrids from Chytridiales and Neocallimasticales
5
3. REVIEW OF LITERATURE: 3.3 Current status in the taxonomy of Entomophthorales
/NAGAHAMA et al. 1995, JENSEN et al. 1998/. Recently, the family Neozygitaceae has not
been distinguished by BAŁAZY /1993/ and all the Neozygites species have been organized by
him within the family Entomophthoraceae. The families within the order Entomophthorales
are distinguished primarily on the basis of nuclear characters /HUMBER 1981, 1984, 1987,
1989, BAŁAZY 1993/. The families Entomophthoraceae and Neozygitaceae consist
exclusively of insect and mite pathogens, while only a few insect pathogenic species are
found in Ancylistaceae.
Figure 1. Systematic position of Entomophthorales within the Kingdom of Fungi
KINGDOM
Fungi
PHYLUM
Chytridiomycota Zygomycota Ascomycota Basidiomycota
CLASS Trichomycetes Zygomycetes
ORDER Mucorales Entomophthorales
Ancylistaceae Entomophthoraceae
FAMILY
Neozygitaceae Completoriaceae
Meristacraceae Basidiobolaceae
________________________________________________________________________
Accepted by the Dictionary of Fungi /HAWKSWORTH et al. 1995/
Main characters for generic classification include a mode of discharge of primary conidia,
a structure of conidial wall, a number of nuclei per conidium, a shape of primary and
secondary conidia, a mode of formation of secondary conidia, a presence of cystidia and
rhizoids, and finally a pathobiology /HUMBER 1981, 1987, 1989, KELLER 1987a, 1991, 1999/.
However, which groups of characters define genera within the Entomophthorales has not
always been of general acceptance. REMAUDIÈRE and KELLER /1980/ said that the shape of
primary conidia should be the main criterion for generic classification, while HUMBER /1981/
recommended that primary conidium shape should be secondary to nucleation, branching of
conidiophores and mode of discharge of primary conidia. Recent results of molecular analyses
/HAJEK et al. 2003/ support the importance of primary conidial shape in generic grouping. A
total of 14 entomopathogenic genera are present within the three families. In addition, there is
one more genus in the family Basidiobolaceae. EILENBERG /2002/ listed altogether 236
species within the 14 genera. Though, the number of species can be disputed. There is a
plenty of insufficiently described species and several species have been described several
times resulting in many synonyms.
Since the first entomophthoralean species was described there arose much confusion and
equivocation as regard the nomenclatural and taxonomic issues and many of them still have
6
3. REVIEW OF LITERATURE: 3.3 Current status in the taxonomy of Entomophthorales
Figure 2. Different classifications of arthropod-pathogenic fungi in the order Entomophthorales

BEN-ZE’EV and BAŁAZY 1993 KELLER 1987, 1991, HUMBER 1989, 1997
KENNETH 1982a,b 1999
Ancylistaceae Ancylistaceae Ancylistaceae Ancylistaceae
Conidiobolus Conidiobolus Conidiobolus  Conidiobolus 
subg. Conidiobolus subg. Conidiobolus
subg. Delacroixia subg. Delacroixia
subg. Capillidium subg. Capillidium
Entomophthoraceae Entomophthoraceae Entomophthoraceae Entomophthoraceae
Entomophaga Entomophaga Entomophaga Entomophaga
Batkoa Batkoa Batkoa 
Entomophthora Entomophthora Entomophthora Entomophthora
Eryniopsis  Eryniopsis  Eryniopsis 
Massospora Massospora Massospora Massospora
Orthomyces 
Strongwellsea Strongwellsea Strongwellsea Strongwellsea
Erynia  Zoophthora   
subg. Zoophthora subg. Zoophthora Zoophthora Zoophthora
subg. Neopandora subg. Neopandora Erynia s.l. Pandora
subg. Erynia subg. Erynia Erynia
subg. Furia subg. Furia Furia
Tarichium  Tarichium  Tarichium  Tarichium 
(form genus) (form genus) (form genus) (form genus)
Triplosporium  Neozygites  Neozygitaceae Neozygitaceae

Neozygites Neozygites
Entomophthora 
(nomina provisoria) Basidiobolaceae  Basidiobolaceae 
Basidiobolus Basidiobolus
___________________________________________________________________________
 KELLER /1987, 1999/ and HUMBER /1989, 1997/ do not split Conidiobolus into three subgenera,
 New genus described in 1989 /HUMBER 1989/,
 New genus described in 1984 /HUMBER 1984/,
 New genus described in 1998 /STEINKRAUS et al. 1998b/,
 Keller and Humber do not use Zoophthora as a genus name for all uninucleate genera; Humber elevated all
the subgenera to the generic rank,
 BEN-ZE’EV and KENNETH /1982a/ prefer the generic name Triplosporium to the name Neozygites; BAŁAZY
/1993/ left Neozygites genus within the family Entomophthoraceae,
 A form genus for fungi with unknown conidial stage,
 A provisional group of insufficiently described species,
 Recent phylogenetic studies showed that the genus did not belong to Entomophthorales
not been solved completely. However, the classification in Entomophthorales has over the
years passed a great interest of many taxonomists and during the last few years the taxonomy
has undergone a revision and changed dramatically. Though at the beginning of the
investigation on Entomophthorales these fungi were first classified into the class of
Basidiomycetes and ten years later Nowakowski correctly classified them into the family
Entomophthoraceae within the group Zygomycetes /BAŁAZY 1993/. Four basic monograph
7
3. REVIEW OF LITERATURE: 3.4 Aphidophagous Entomophthorales
of Entomophthoraceae were published in the second half of 19th century and at the beginning
of the 20th century /FRESENIUS 1858, NOWAKOWSKI 1883, THAXTER 1888, LAKON 1919/. The
Thaxter’s monograph became the most influential and had many followers. THAXTER /1888/
employed one genus Empusa for all insect attacking species and the genus comprised three
subgenera: Empusa, Entomophthora and Triplosporium. Thaxter further accepted the genera
Basidiobolus, Conidiobolus, Completoria, and Massospora as belonging into
Entomophthoraceae. Lakon’s classification /LAKON 1919/ was limited to the insect-
pathogenic species of the family and was basically an emended version of Nowakowski’s
system /BEN-ZE’EV and KENNETH 1982a/. Essentially, until 1963 only the genera
Entomophthora and Massospora were recognised for insect-pathogenic species /MACLEOD
1963/. Few years later a further genus, Strongwellsea, was described /BATKO and WEISER
1965/. While the genera Massospora and Strongwellsea comprised only few well-defined
species, Entomophthora consisted of nearly 100 species morphologically rather diverse.
BATKO /1964b,c,d,e, 1966b/, aware of the heterogeneity within the genus, was the first who
laid the basis for a new classification by splitting this large genus into several genera. Since
his system had certain abnormalities and shortcomings, it was not accepted. Consequently,
REMAUDIÈRE and HENNEBERT /1980/, REMAUDIÈRE and KELLER /1980/ and HUMBER /1981,
1984/ revised the Batko’s system and defined seven genera. These corrections found a wide
acceptance. Simultaneously, main morphological and taxonomic criteria used in
Entomophthorales were revised and evaluated /HUMBER 1981, BEN-ZE’EV and KENNETH
1982a/. Later, more genera were proposed by splitting the genera Entomophaga, Erynia
/HUMBER 1989/ and Neozygites /BEN-ZE’EV et al. 1987/. The split of Neozygites into
Neozygites and Thaxterosporium was not justified and recently has been refused /KELLER
1997/. Whereas the split of Entomophaga into Entomophaga and Batkoa has been accepted
/BAŁAZY 1993, KELLER 1999/, the split of Erynia into Erynia, Furia and Pandora has been
considered as premature and not accepted /KELLER 1991, 1999/ or the split has been accepted
but the genera has been treated as subgenera of Zoophthora /BAŁAZY 1993/.
At present four systems of Entomophthorales classification are normally used, referred as
Ben-Ze’ev and Kenneth’s classification, Bałazy’s classification, Keller’s classification and
Humber’s classification (Figure 2). In this work the system proposed by HUMBER /1989,
1997/ is followed.
3.4 APHIDOPHAGOUS ENTOMOPHTHORALES

This chapter contains a complete list of entomophthoralean species, which have been
recorded from aphid hosts in literature. Altogether 31 pathogenic fungi organised within four
families are listed herein. The fungi are arranged into families and a description of each
species is focused on a nomenclature genesis and history, a distribution in the world, host
specificity, a culture in vitro, and possible prospects for the use in the biological control.
Family: Entomophthoraceae Nowakowski, Bot. Ztg. 34 (14): 216-222, 1877

Genus: Pandora Humber, Mycotaxon 34: 451-453, 1989
Syn.: Zoophthora subgenus Pandora Batko (pro parte), Acta Mycol. 2: 18-19, 1966b (as described
but excluding the designated type, Entomophthora aphidis Hoffmann in Fresenius, which is a
species of Zoophthora sensu stricto)
Erynia subgenus Neopandora Ben-Ze’ev et Kenneth, Mycotaxon 14: 460-462, 1982b
Zoophthora subgenus Neopandora Ben-Ze’ev et Kenneth (sensu Bałazy 1993), Flora of Poland,
Fungi (Mycota) 24: 171-172, 1993
8
The genus was designated by HUMBER /1989/ after a critical revision of four Batkoan
subgenera of Zoophthora genus. Involving primary and secondary generic characters as well
as a general pathobiology of the fungi, the Batkoan subgenus Pandora was redescribed and
raised to a generic rank. Originally, 16 species were included in this genus and it was typified
by Pandora neoaphidis (Remaudière et Hennebert) Humber. Recently, however, BAŁAZY
/1993/ suggested to maintain the subgenus Neopandora Ben-Ze’ev et Kenneth as one of four
subgenera of the genus Zoophthora Batko. The subgenus Neopandora was formerly proposed
by BEN-ZE’EV and KENNETH /1982b/ as a subgenus of the genus Erynia Nowakowski, emend.
Humber et Ben-Ze’ev. Further, Bałazy synonymized one species with a new one, excluded 2
species and added another 10 new species to the subgenus. Out of 24 species described up to
now four are pathogenic to aphids. Three species are obligate pathogens of aphids while one
is a pathogen of plant- and leaf-hoppers but can artificially attack aphids, as well.
1. Pandora neoaphidis (Remaudière et Hennebert) Humber, Mycotaxon 34: 452, 1989

Syn.: Entomophthora aphidis Hoffman in Fresenius sensu Thaxter, Mem. Boston Soc. nat. Hist. 4:
152-189, 1888
Bas.: Erynia subgenus Neopandora neoaphidis Remaudière et Hennebert ex Ben-Ze’ev et Kenneth,
Mycotaxon 14: 461-462, 1982b
Erynia neoaphidis Remaudière et Hennebert, Mycotaxon 11: 307-312, 1980
Zoophthora subgenus Neopandora neoaphidis (Remaudière et Hennebert) Bałazy, Flora of Poland,
Fungi (Mycota) 24: 175-176, 1993
The species was originally discovered by THAXTER /1888/ in the USA and the fungus was
being incorrectly interchanged with a taxon Entomophthora aphidis Hoffman in Fresenius
(today known as a basionym of species Zoophthora aphidis (Hoffman in Fresenius) Batko)
for nearly 100 years /REMAUDIÈRE and HENNEBERT 1980/. THAXTER /1888/, in his influential
monograph, applied the name Empusa (Entomophthora) aphidis to the fungal pathogen of
aphids which produced conidia and whose resting spore stage might be assumed to exist but
were not found. Besides an incorrectly application of the generic name Empusa to the fungus
(this generic name, applied to the fungal pathogens by COHN /1855/ for the first time, was
properly rejected by FRESENIUS /1856/, since it had been already used for orchids by Lindley,
hence he used a generic name Entomophthora), the mycologist also incorrectly treated his
fungus as synonymous with Entomophthora aphidis Hoffman in Fresenius, although the
primary description of this organism by Hoffmann was originally based on brown roughened
resting spores and a conidial stage of this species was not found or described /REMAUDIÈRE
and HENNEBERT 1980/. BATKO /1966b/ was unaware that this name had been misapplied for
yet undescribed fungus and typified his subgenus Pandora (in the genus Zoophthora) with
Entomophthora aphidis Hoffman in Fresenius as Zoophthora (Pandora) aphidis (Hoffman in
Fresenius) Batko.
After nearly 100 years REMAUDIÈRE and HENNEBERT /1980/ successfully rediscovered
Hoffman’s fungus and demonstrated that Thaxter misapplied the Hoffman’s specific name.
The taxonomists used the presence of capilliconidia in the rediscovered Hoffmann’s E.
aphidis and not in the Thaxter’s fungus to place these two fungi into separate genera. The two
different species were redescribed, the Thaxter’s misapplied species under a new name as
Erynia neoaphidis Remaudière et Hennebert, and the Hoffmann’s E. aphidis as Zoophthora
aphidis (Hoffman in Fresenius) Batko. HUMBER and BEN-ZE’EV /1981/ emended a sense of
genus Erynia Nowakowski, rejected Zoophthora as a separate genus for species forming
capilliconidia, and placed both those species in the same genus as Erynia neoaphidis
Remaudière et Hennebert and Erynia aphidis (Hoffman in Fresenius) Humber et Ben-Ze’ev.
Consequently, the description of genus Erynia Nowakowski emend. Humber et Ben-Ze’ev
9
was emended again and all recognized species of the genus were reclassified into four
subgenera. E. neoaphidis was transferred into new subgenus Neopandora /BEN-ZE’EV and
KENNETH 1982b/. HUMBER /1989/ revised familial and generic criteria of Entomophthorales
and elevated subgenera of Erynia genus to a generic rank. E. (Neopandora) neoaphidis was
allocated to new genus Pandora. Finally, BAŁAZY /1993/ reclassified again the species into
subgenus Neopandora Ben-Ze’ev et Kenneth of genus Zoophthora Batko as Zoophthora
(Neopandora) neoaphidis (Remaudière et Hennebert) Bałazy.
As for morphological and physiological properties, the species exhibits certain degree of
variability. For instance, conidia collected from Brevicoryne brassicae (Linné) were distinctly
broader that conidia from other P. neoaphidis strains and further all attempts to isolate the
fungus from the aphid failed /KELLER 1991/. This taxon is usually considered a species
complex /HUMBER 1983/ though there is no evidence for this /HUMBER 1990/. BAŁAZY /1993/
also pointed out that the taxon, in the sense referred in current taxonomic literature, is a
species complex.
P. neoaphidis is a common aphid pathogen with a distribution all over the world although
it is less frequent in tropical regions. In Poland the fungus was recorded from 23 aphid species
in many localities all over the country. A period of pathogen’s appearance in the nature is
usually from the second half of May till the first frost in the autumn /BAŁAZY 1993/. In
Switzerland the species was discovered in number of localities north and south of the Alps as
well as in some alpine valleys up to a level of about 2000 m above sea level. P. neoaphidis
was found between April and December and regularly caused epizootics among aphid
populations in field crops; the pathogen was identified from 23 aphid species in the country
/KELLER 1991/. It is considered the most important aphid pathogenic fungus in Switzerland
/KELLER and SUTER 1980/. Taxon Entomophthora aphidis was recorded from 92 aphid
species in France /THOIZON 1970/, however, the list of records was prepared before the
revision of the taxon by REMAUDIÈRE and HENNEBERT /1980/ and the erection of new species
E. neoaphidis. P. neoaphidis is a pathogen of many aphid species generally considered to be
the most important agent responsible for fungal epizootics in host populations /e.g.
LEATHERDALE 1970, THOIZON 1970, MILNER 1981a, MIETKIEWSKI and VAN DER GEEST 1985,
HUMBER 1990, KELLER 1991, BAŁAZY 1993, STEENBERG and EILENBERG 1995, EILENBERG
2002/. This species is regularly recorded as a principal aphid enemy within colonies of cereal
aphids /e.g. DEAN and WILDING 1971, DEDRYVER 1978, 1981, 1983, COREMANS-PELSENEER
et al. 1983, PAPIEROK and HAVUKKALA 1986, OZINO et al. 1988, FENG et al. 1990,1992a,
FENG and NOWIERSKI 1991, STEENBERG and EILENBERG 1995, BASKY and HOPPER 2000,
HATTING et al. 1999a, 2000, ŠTALMACHOVÁ and CAGÁŇ 2000, HEMMATI et al. 2001b,
ABDEL-MALLEK et al. 2003, ABDEL-RAHMAN and ALI 2003/, pea aphid /e.g. WILDING 1975,
MILNER 1982, 1985, PICKERING et al. 1989a, PICKERING and GUTIERREZ 1991, BEHRENS
1993, CAGÁŇ and BARTA 2001, MAJCHROVICZ 2001/, black bean aphid /e.g. GUSTAFSSON
1969, MAJCHROWICZ 1979, WILDING and PERRY 1980/, cabbage aphid /LOWE 1963, 1968a,b,
SIVČEV 1991, 1992/, black cherry aphid /KLINGEN and JAASTAD 2003/, or green peach aphid
/e.g. SHANDS et al. 1972, ELKASSABANY et al. 1992, KISH et al. 1994, MCLEOD et al. 1998,
MCLEOD and STEINKRAUS 1999, DARA and SEMTNER 2001/.
The fungus can be easily isolated and cultivated on solid media based on Sabouraud’s
dextrose agar enriched with cow’s milk and egg yolk /KELLER 1991/ as well as in liquid
media /LATGÉ et al 1983, LI et al. 1993/.
P. neoaphidis is regarded to have a great potential as an agent for biological control of
aphid populations and several attempts have been made to introduce the fungus into aphid
populations in laboratory or field /e.g. WILDING 1981, LATTEUR and GODEFROID 1983,
WILDING et al. 1986a,b, 1990, SHAH et al. 2000a/. However, a mass production, an inoculum
10
storage, and an inoculum formulation of the fungus are still obstacles of its commercial use
/DEDRYVER 1981, LATGÉ et al. 1983, SHAH et al. 1998, 1999, 2000b).
2. Pandora delphacis (Hori) Humber, Mycotaxon 34: 452, 1989

Bas.: Entomophthora delphacis Hori, Entomol. Mag. (Tokyo) 3: 81, 1906
Erynia delphacis (Hori) Humber, Mycotaxon 13: 212, 1981
Zoophthora subgenus Neopandora delphacis (Hori) Bałazy, Flora of Poland, Fungi (Mycota) 24:
183-185, 1993
The species Pandora delphacis was for the first time described (as Entomophthora
delphacis Hori) and isolated from its hosts Nilaparvata lugens (Stål) and Sogatella furcifera
Horváth (Homoptera: Delphacidae) in Japan at the beginning of the last century /HORI 1906/.
P. delphacis bears a strong morphological resemblance to P. neoaphidis but differs notably
in the complete lack of rhizoids on any of its hosts. Further, isolates of P. delphacis grow
considerably more rapidly on a wider variety of nutritionally simple or complex media and
sporulate more intensively over a longer period of time than any cultures of P. neoaphidis.
The species differ also in a range of their hosts and in mobility of specific enzymes /HUMBER
1981, BEN-ZE’EV and KENNETH 1982b, MILNER et al. 1983, HUMBER 1990/. REMAUDIÈRE
and HENNEBERT /1980/ considered the differences of growth in vitro between those two
species to be insignificant and regarded Erynia delphacis (= P. delphacis) as a nomen dubium.
This was fully accepted by REMAUDIÈRE and KELLER /1980/, as well. P. delphacis was not
mentioned by Batko in any of his papers /BATKO 1964b,c, 1966b/ and this species was
properly allocated to the genus Erynia (subgenus Erynia) by BEN-ZE’EV and KENNETH
/1982b/. HUMBER /1989/ in the sense of his emendations placed the fungus in the genus
Pandora.
At present the fungus is primarily known as a pathogen of leaf- and planthoppers in rice
paddies in the Southeast Asia and Indonesia where the pathogen causes regularly epizootics in
host populations /LI 1988, HOLDOM et al. 1989, NARAYANASAMY et al. 1992, AMBETHGAR
1996, MATSUI et al. 1998). However, recently the pathogen was also recorded from
Spissistilus festinus Say, a member of the family Membracidae (Homoptera), in the North
America /MILLER and HARPER 1987/. Several strains of the fungus were isolated from
Empoasca fabae (Harris) (Homoptera: Cicadellidae) in the USA and cicadellids in Brazil
/HUMBER 2001/. BAŁAZY /1993/ found a fungus of identical morphology on a specimen of
Auchenorrhyncha (= Cicadoidea) in Germany on a grass stem in a meadow. The most recent
information says that P. delphacis is capable to attack besides the homopterans some species
of the order Lepidoptera. There is a record of a successful isolation of P. delphacis from non-
homopteran species, Mamestra brassicae (Linné) (Lepidoptera: Noctuidae), in Japan
/HUMBER 2001/.
The fungus is able to infect some aphid species in laboratory, as well (SHIMAZU 1977,
MILNER et al. 1983, XU and FENG 2000, 2002). Though it has never been observed infecting
aphids in nature, results of laboratory experiments exhibited even greater pathogenicity of P.
delphacis for Myzus persicae Sulzer than isolates of P. neoaphidis (XU and FENG 2002). The
results suggest that P. delphacis has a potential as valuable fungal agent for control of aphids.
3. Pandora nouryi (Remaudière et Hennebert) Humber, Mycotaxon 34: 453, 1989

Bas.: Erynia nouryi Remaudière et Hennebert, Mycotaxon 11: 313-316, 1980
Zoophthora nouryi (Remaudière et Hennebert) Ben-Ze’ev et Kenneth, Phytoparasitica 9: 40-41,
1981
Erynia subgenus Neopandora nouryi Remaudière et Hennebert ex Ben-Ze’ev et Kenneth,
Mycotaxon 14: 462, 1982b
11
Zoophthora subgenus Neopandora nouryi (Remaudière et Hennebert) Ben-Ze’ev et Kenneth sensu

Bałazy, Flora of Poland. Fungi (Mycota) 24: 195, 1993
Syn.: Erynia exitialis Hall et Dunn sensu Gustafsson 1965, Lantburkshögskolans annaler 32: 102-212,
1965
Until 1980 the species was not separated from collections identified as “Entomophthora
aphidis” in the sense of Pandora neoaphidis or as “Entomophthora exitialis”. REMAUDIÈRE
and HENNEBERT /1980/ revised these groups of pathogens and erected a new species Erynia
neoaphidis to include collections previously referred to as Entomophthora aphidis Hoffman in
Fresenius sensu Thaxter. Entomophthora exitialis Hall et Dunn /HALL and DUNN 1957/ was
regarded as a nomen confusum and consequently rejected /REMAUDIÈRE and HENNEBERT
1980/. Those collections of E. exitialis having ovoid primary spores less than 18 μm in length
and forming resting spores (i.e. Entomophthora exitialis Hall et Dunn sensu Gustafsson) were
renamed Erynia nouryi Remaudière et Hennebert. Subsequently, this revision was accepted
by other taxonomists /REMAUDIÈRE and KELLER 1980, HUMBER and BEN- ZE’EV 1981,
MILNER 1981a, MILNER et al. 1983/. During next years the taxon was reclassified and
removed to genus Pandora /HUMBER 1989/ or Zoophthora subgenus Neopandora /BAŁAZY
1993/.
P. nouryi is known only from Europe mainly on the root dwelling Pemphigus bursarius
Linné, but also from Aphis fabae Scopoli and Aphis umbrella Börner. The first aphid species
is considered as the typical host /GUSTAFSSON 1965, MILNER 1981a, BAŁAZY 1993/. In
Poland the fungus was identified from P. bursarius and Pemphigus phenax Börner /BAŁAZY
1993, MAJCHROWICZ 1999/. In general, P. nouryi is a rare entomopathogen in aphid
populations /REMAUDIÈRE and HENNEBERT 1980/. HUMBER /2001/ states isolates of P. nouryi
in “USDA-ARS Collection of entomopathogenic fungal cultures” with origin from France (on
Therioaphis maculata Buckton) and from outside of Europe in the USA (on aphids from
potatoes), or Australia (on Acyrthosiphon kondoi Shinji).
The fungus develops in cultures on media containing Sabouraud’s agar, egg yolk and milk
/GUSTAFSSON 1965, REMAUDIÈRE and HENNEBERT 1980, MILNER et al. 1983, BAŁAZY 1993/.
4. Pandora kondoiensis (Milner) Humber, Mycotaxon 34: 453, 1989

Bas.: Erynia kondoiensis (Milner), in Milner, Mahon et Brown, Austral. J. Bot. 31: 183, 1983
Zoophthora subgenus Neopandora kondoiensis (Milner) Bałazy, Flora of Poland, Fungi (Mycota)
24: 195-196, 1993
The pathogen was described from aphid Acyrthosiphon kondoi Shinji (a type host for the
species) in Australia in 1977 by MILNER et al. /1983/ and named Erynia kondoiensis Milner.
The taxon was successfully separated from an aphidicolous group closely related to P.
neoaphidis. Apart from differences in the morphology and the fatty acid content it may be
distinguished from P. neoaphidis, P. nouryi and P. delphacis by specific enzyme mobility. P.
kondoiensis is very similar to P. neoaphidis, but has smaller primary spores and the
mycelium contains a much higher proportion of C 12:0 fatty acids. In Australia P. kondoiensis
causes epizootics in A. kondoi populations, but it was rarely observed in the sympatric
Acyrthosiphon pisum Harris. Conversely, A. pisum was frequently infected with P.
neoaphidis. It was suggested that the fungus could usefully be imported into New Zealand and
the USA for biological control of A. kondoi. Further aphid species, A. pisum, Therioaphis
trifolii (Monell), Hyperomyzus lactucae Linné and M. persicae, were also infected artificially
/MILNER et al. 1983, BAŁAZY 1993/.
Outside of Australia the pathogen was described in China from M. persicae where it
occurred epizootically in the aphid population on tobacco /FAN et al. 1991/.
12
New species, Entomophthora terrestris Gres et Kovaľ, was described from Pemphigus
fuscicornis Koch in the Ukraine /Gres and Kovaľ 1982/. This incompletely described species
is probably synonymous with P. nouryi /Keller 2004, person. comm./.
Cultures of the fungus can be established on media containing Sabouraud’s agar, egg yolk
and milk /MILNER et al. 1983/.
Genus: Erynia (Nowakowski ex Batko) Remaudière et Hennebert emend. Humber,

Mycotaxon 34: 448-449, 1989
Syn.: Zoophthora subgenus Erynia Nowakowski ex Batko, Acta Mycol. 2: 18, 1966b
Erynia (Nowakowski ex Batko) Remaudière et Hennebert, Mycotaxon 11: 301, 1980
Erynia subgenus Erynia Batko ex Ben-Ze’ev et Kenneth, Mycotaxon 14: 459, 1982
This generic name was first published in a report of meeting of Polish physicians and
biologists by Nowakowski in 1881. Nowakowski later rejected this generic name in favour of
Brefeld’s Entomophthora /HUMBER 1981, 1989/. BATKO /1964b, 1966b/ believing that
Nowakowski’s later disuse of Erynia removed this name from consideration in matters of
nomenclatural priority and proposed the genus Zoophthora consisted of four subgenera with
Erynia as one of them. REMAUDIÈRE and HENNEBERT /1980/ redefined Erynia Nowakowski,
altering both Nowakowski’s generic description and Batko’s subgeneric one. Consequently,
the description of Erynia Nowakowski was again emended /HUMBER and BEN-ZE’EV 1981,
BEN-ZE’EV and KENNETH 1982b/. Whereas HUMBER /1989/ concluded that genus Erynia
Nowakowski is a nomen nudum and elevated all four Batkoan subgenera of Zoophthora to
generic rank, KELLER /1991/ maintained the systematic arrangement proposed by
REMAUDIÈRE and HENNEBERT /1980/. On the other hand BAŁAZY /1993/ supported to
preserve the Batko’s Zoophthora genus with the subgenera based upon BEN-ZE’EV and
KENNETH /1982b/. At present the genus in the sense of Humber includes altogether 12
different species /HUMBER 1989, BAŁAZY 1993/.
5. Erynia erinacea (Ben-Ze’ev et Kenneth) Remaudière et Hennebert, Mycotaxon 11:

302, 1980
Bas.: Zoophthora erinacea Ben-Ze’ev et Kenneth, Mycotaxon 10: 219-232, 1979
Erynia subgenus Erynia erinacea (Ben-Ze’ev et Kenneth) Remaudière et Hennebert ex Ben-Ze’ev
et Kenneth, Mycotaxon 14: 459, 1982b
Zoophthora subgenus Erynia erinacea Ben-Ze’ev et Kenneth sensu Bałazy, Flora of Poland, Fungi
(Mycota) 24: 155-156, 1993
The taxon was described from Aphis craccivora Koch on alfalfa in Israel in 1977 and 1978
and was named Zoophthora erinacea Ben-Ze’ev et Kenneth. It caused different degrees of
mortality in populations of Aphis umbrella Börner and M. persicae inhabiting Malva sp. and
in Aphis fabae populations in several localities. Further, aphid species Aphis spiraecola Patch
was successfully inoculated by this pathogen in laboratory /BEN-ZE’EV and KENNETH 1979/.
The fungus has not yet been recorded from any other country.
According to the morphological features of the taxon HUMBER /1989/ allocated it to the
genus Erynia and BAŁAZY /1993/ to the genus Zoophthora subgenus Erynia.
Any attempts to isolate and cultivate the fungus on egg yolk medium and several rich agar
media failed. However, it started to grow very slowly on egg-yolk what sustained the hope of
future isolation /BEN-ZE’EV and KENNETH 1979/.
6. Erynia conica (Nowakowski) Remaudière et Hennebert, Mycotaxon 11: 302, 1980
13
Bas.: Entomophthora conica Nowakowski, Pam. Akad. Umiej. Kraków, Wydz. Mat.-Przyr. 8: 155,
1883
Empusa subgenus Entomophthora conica Nowakowski ex Thaxter, Mem. Boston Soc. Nat. Hist. 4:
186-187, 1888
Zoophthora conica (Nowakowski) Batko, Bull. Acad. Pol. Sci., Ser. Sci. Biol. 12: 404-405, 1964c
Zoophthora subgenus Erynia conica (Nowakowski) Batko ex Batko, Acta Mycol. 2: 18, 1966b
Erynia subgenus Erynia conica (Nowakowski) Remaudière et Hennebert ex Ben-Ze’ev et Kenneth,
Zoophthora subgenus Erynia conica (Nowakowski) Batko in Bałazy, Flora of Poland, Fungi
(Mycota) 24: 154, 1993
E. conica was described by Nowakowski at the end of 19th century /HUMBER 1989/.
Nowadays this fungus is known as a common pathogen on members of the families Culicidae
and Chironomidae around lakes, ponds and at river or brook banks just over the water surface
in Europe, North America, Asia and Australia. In Poland it was observed from May through
vegetation season in numbers of localities /BAŁAZY 1993/. In Switzerland E. conica infected
unidentified specimens of Chironomidae from mid-August till end of October /KELLER 1991/.
In the literature, there is just one record of E. conica from aphids until now. Pathogen
Entomophthora conica was observed in France on five aphid species: Aulacorthum solani
Kaltenbach, Brachycaudus klugkisti Börner, Myzus ascalonicus Doncaster, Myzus cerasi
(Fabricius) and Paramyzus heraclei Börner /THOIZON 1970/. However, those determinations
are at least dubious since no other authorities have yet confirmed aphidophagous properties of
the fungus. WATERHOUSE and BRADY /1982/ in their “Key to the species of Entomophthora s.
lato” also questioned about the ability of E. conica to infect aphids.
Genus: Zoophthora (Batko) Remaudière et Hennebert emend., Mycotaxon 11: 301, 1980
Syn.: Zoophthora subgenus Zoophthora Batko, Acta Mycol. 2: 16-18, 1966b
Zoophthora subgenus Zoophthora Batko (sensu Bałazy 1993), Flora of Poland, Fungi (Mycota) 24:
147-149, 1993
Erynia subgenus Zoophthora Ben-Ze’ev et Kenneth, Mycotaxon 14: 466-469, 1982b
Entomophthora subgenus Lophorhiza Giard, Bull. Sci. France Belgique 19: 303-304, 1888
The genus Zoophthora with its four subgenera was erected by BATKO /1964b, 1966b/ and
later revised by REMAUDIÈRE and HENNEBERT /1980/. HUMBER and BEN-ZE’EV /1981/
emended a sense of genus Erynia Nowakowski and rejected a separation of Zoophthora Batko
(sensu Remaudière et Hennebert) from the genus Erynia, which had been based on the
presence of capilliconidia in the former one or on any other criteria. The genus Zoophthora
was rejected as later synonym of the genus Erynia and the species of Zoophthora were placed
in Erynia. On the other hand an opinion to preserve the Batkoan intrageneric differentiation
was practically adopted with some corrections by BEN-ZE’EV and KENNETH /1982b/. The
mycologists, however, accepted a priority of genus Erynia Nowakowski over Zoophthora
Batko and transferred the genus Zoophthora Batko as subgenus to Erynia Nowakowski
emend. Humber et Ben-Ze’ev. Several years later HUMBER /1989/ came to conclusion that the
name Erynia Nowakowski is a nomen nudum and cannot be applied as a valid generic name.
Simultaneously, he elevated all four Batkoan subgenera to the generic rank. In this sense the
subgenus Zoophthora was raised to genera as Zoophthora Batko with 13 species and
Zoophthora radicans (Brefeld) Batko was accepted as a type species of the genus. BAŁAZY
/1993/ in his monograph emphasised to be reasonable to maintain the genus Zoophthora sensu
BATKO /1964b/ with its four subgenera emended by BEN-ZE’EV and KENNETH /1982b/ and
described further14 new species within the subgenus Zoophthora (in the genus Zoophthora).
14
At present the genus encompasses a homogenous group of 30 species, all of which are
pathogenic for insects, with 7 ones pathogenic for aphids. Out of the 7 pathogens two are
rather wide polyphagous organisms, while remaining ones are obligatorily aphidophagous
/HUMBER 1989, KELLER 1991, BAŁAZY 1993/.
7. Zoophthora aphidis (Hoffman in Fresenius) Remaudière et Hennebert, Mycotaxon 11:

285-294, 1980
Bas.: nec Zoophthora aphidis (Hoffman in Fresenius) Batko, Bull. Acad. Pol. Sci., Ser. Sci. Biol. 12:
323-324, 1964b
Entomophthora aphidis Hoffman in Fresenius, Abhandl. Senckenberg. Naturforsch. Gesellsch. 2:
208, 1858
Tarichium aphidis (Hoffman in Fresenius) Cohn, Beitr. Biol. Pflanz. 1: 84, 1870
Erynia aphidis (Hoffman in Fresenius) Humber et Ben-Ze’ev, Mycotaxon 13: 509, 1981
Erynia subgenus Zoophthora aphidis (Hoffman in Fresenius) Humber et Ben-Ze’ev ex Ben-Ze’ev
et Kenneth, Mycotaxon 14: 467, 1982b
Zoophthora subgenus Zoophthora aphidis (Hoffman in Fresenius) Remaudière et Hennebert (sensu
Bałazy 1993), Flora of Poland, Fungi (Mycota) 24: 211-216, 1993
The species was for the first time described under a name of Entomophthora aphidis
Hoffman in Fresenius only from its brown spherical resting spores observed inside of
cadavers of Anoecia corni (Fresenius) in France. A conidial stage of this species was not
found or described by Hoffman /REMAUDIÈRE and HENNEBERT 1980/. THAXTER /1888/ used a
name of Empusa (Entomophthora) aphidis to the most frequent fungal pathogen of aphids
which forms conidia, but resting spores have not been observed yet. So, the name
Entomophthora aphidis Hoffman in Fresenius sensu Thaxter was till 1980 incorrectly applied
to another, undescribed fungus, known at present under the name Pandora neoaphidis
(Remaudière et Hennebert) Humber. In 1980 REMAUDIÈRE and HENNEBERT /1980/
rediscovered Hoffman’s fungus and confirmed that Thaxter had misapplied the Hoffman’s
specific name for a different species. The problem of nomenclatural status of this species was
solved by describing a new taxon (E. neoaphidis) as well as by redescribing the Hoffman’s
fungus as Zoophthora aphidis (Hoffman in Fresenius) Batko. During the next few years a
taxonomic position of Z. aphidis was changed by some authors /HUMBER and BEN-ZE’EV
1981, BEN-ZE’EV and KENNETH 1982b/, or was maintained /REMAUDIÈRE and KELLER 1980,
HUMBER 1989, KELLER 1991/. BAŁAZY /1993/ proposed to refuse the name of Z. aphidis
(Hoffman in Fresenius) sensu Batko for this taxon since BATKO /1966b/ typified his subgenus
Pandora with the name of Z. aphidis. Bałazy ascribed a correct sense of this taxon to
REMAUDIÈRE and HENNEBERT /1980/.
E. aphidis Hoffman in Fresenius was also erroneously applied for other aphid pathogens:
Zoophthora canadensis (MacLeod, Tyrrell et Soper) Remaudière et Hennebert, as a result of
misidentification, and Pandora nouryi (Remaudière et Hennebert) Humber, which was not
distinguished from Pandora neoaphidis (Remaudière et Hennebert) Humber. The problem
was solved by REMAUDIÈRE and HENNEBERT /1980/.
The pathogen is distributed in Western and Central Europe on aphids of the genus Anoecia
sp. infesting leaves of Cornus sp. in autumn. The fungus was observed on several localities in
Poland in 1933 and 1980 /BAŁAZY 1993/, however, attempts to find this pathogen in
populations of Anoecia sp. on Cornus sanguinea Linné in Poland, Romania, Eastern France or
Germany have been unsuccessful for last 2 decades /BAŁAZY 1993/. In Switzerland the
pathogen was identified from two aphid species Anoecia corni (Fresenius) and
Rhopalosiphum padi Linné. Z. aphidis was found between the end of September and the end
of October in populations of sexuales, epizootics and high mortalities were sometimes
observed, as well /KELLER 1991/. The author also noticed that aphids with sporulating fungus
15
are fixed by rhizoids to the underside of leaves, whereas aphids containing resting spores are
mainly found around the buds in the leaf shoulders or sometimes along the main veins of the
leaves. In general, the species is considered a rather uncommon pathogen with distribution in
Europe /MILNER 1981a/ and till 1991 regarded as monophagous for Anoecia sp. Z. aphidis has
probably a low value for biological control and no efforts to exploit the fungus in this way
have been published. However, the fungus is easy to isolate and cultivate on Sabouraud’s
agars with yolk and milk /KELLER 1991, BAŁAZY 1993/, but resting spores are not produced
in culture /BAŁAZY 1993/.
8. Zoophthora phalloides Batko, Acta Mycol. 2: 7-13, 1966a

Bas.: Erynia phalloides (Batko) Humber et Ben-Ze’ev, Mycotaxon 13: 505, 1981
Erynia subgenus Zoophthora phalloides (Batko) Humber et Ben-Ze’ev ex Ben-Ze’ev et Kenneth,
Z. phalloides was first described as a pathogen of aphids in Poland by BATKO /1966a/ and
has since been also recorded in North America, Australia and New Zealand /GLARE et al.
1985/. Batko placed this new taxon to his subgenus Zoophthora belonging to a genus of the
same name. Since the pathogen’s description it has also been treated as a member of genus
Erynia /HUMBER and BEN-ZE’EV 1981/ or subgenus Zoophthora of genus Erynia /BEN-ZE’EV
and KENNETH 1982b/. At present the species is allocated in the genus Zoophthora /HUMBER
1989, KELLER 1991/, which has been elevated to this rank from Batkoan subgenus
Zoophthora by HUMBER /1989/. Morphologically, the species is very similar to other aphid
pathogenic members of Zoophthora such as Z. radicans, Z. aphidis, and Z. occidentalis.
However, some work was done to divide isolates of broadly defined Z. radicans and Z.
phalloides using spore dimensions, host specificity, growth in vitro, or analyses of
isoenzymes and fatty acid composition as distinguishing criteria /GLARE et al. 1987/. The
taxonometric analyses confirmed that these two species are distinct and also supported the
traditional criterion for separating these species, the morphology of primary conidia, as
satisfactory. The authors further divided the Z. phalloides isolates into two groups on the basis
of their geographical origins, Europe or North America. According to BAŁAZY /1993/,
Batko’s incomplete description of the taxon in relation to morphology or illustration of
capilliconidia as well as inconsistencies between measurements originally obtained by Batko
and Bałazy’s ones obtained after re-examination of the holotype material bring confusions
into a reliable identification of the taxon. Since the drawings of capilliconidia of Z. phalloides
and Z. occidentalis by REMAUDIÈRE and HENNEBERT /1980/ did not show any essential
differences and the shape of primary spores appears too weak criterion for species separation
in this rather variable group of fungi, BAŁAZY /1993/ recommended a careful re-examination
of all the accessible materials to solve this problem.
Z. phalloides is an obligate aphidophagous fungus and seems to be widespread but not
frequent. As mentioned above the species was described in Poland from the nettle aphid
Microlophium carnosum (Buckton) /BATKO 1966a/. Until now, it has been observed in the
Central and Western Europe, Israel, the North America and Australia on different aphid
species /BEN-ZE’EV et al. 1984, GLARE et al. 1985/. REMAUDIÈRE et al. /1976, 1981/ reported
the pathogen from aphids in France. In Switzerland the species was collected between June
and October from A. pisum, Macrosiphum rosae Linné, Metopolophium festucae Theobald
and R. padi. It was never found on aphids in annual crops but in meadows and natural sites
/KELLER 1991/. In Denmark, the pathogen likewise infected R. padi. Similarly, it was not
recorded in annual crops (cereals), but in aphid colonies feeding on their winter host /NIELSEN
et al. 2001a/. The authors also pointed out a different yearly life cycle of the pathogen
compared to another entomophthoralean fungi identified on cereal aphids in Denmark. A
16
survey of entomopathogens in aphid populations infesting natural vegetation in France

showed that Z. phalloides is better adapted to cooler spring and autumn conditions and is rare
during hot summer period /REMAUDIÈRE et al. 1981/. GLARE et al. /1986b/ also stressed the
pathogen’s adaptation to cool and moist climates. Records of the pathogen from E. abietinum
in Iceland /AUSTARÅ et al. 1997/ might support the opinion about its preference to cooler
climate.
Fungus can be easily cultivated on artificial media containing Sabouraud’s agar with milk,
egg yolk, and yeast extract /GLARE et al. 1987/.
9. Zoophthora radicans (Brefeld) Batko, Bull. Acad. Pol. Sci., Ser. Sci. Biol. 12: 324,
1964b
Bas.: Empusa radicans Brefeld, Bot. Ztg. 28: 186, 1870
Entomophthora radicans Brefeld, Bot. Ztg. 35: 345, 1877
Erynia radicans (Brefeld) Humber, Ben-Ze’ev et Kenneth in Humber et Ben-Ze’ev, Mycotaxon 13:
509, 1981
Erynia subgenus Zoophthora radicans (Brefeld) Humber, Ben-Ze’ev et Kenneth ex Ben-Ze’ev et
Kenneth, Mycotaxon 14: 467, 1982b
The species Z. radicans was originally described as a pathogen of caterpillar, Pieris

brassicae Linné by BREFELD /1870/ under the name Empusa radicans Brefeld. A similar
species attacking leafhoppers, Entomophthora sphaerosperma, was described by Fresenius in
1858 and redescribed by THAXTER /1888/. Fungi reported for many years under the name E.
sphaerosperma Fresenius from different host species were treated as synonymous with E.
radicans. BATKO /1964b/ and REMAUDIÈRE and HENNEBERT /1980/ concluded E.
sphaerosperma to be an invalid name according to the rules of the International Code of
Botanical Nomenclature thus the first validly applied name for it was Empusa radicans
Brefeld. Many efforts were made to split the many-hosted E. sphaerosperma into more
consistent species or subspecies. TURIAN /1957/ proposed 2 new subspecies: E.
sphaerosperma subspecies cicadelliphaga attacking cicadas and subspecies elateridiphaga
attacking Elateridae (Coleoptera). BATKO /1964b/ split E. sphaerosperma into 2 new
combinations: Zoophthora radicans (Brefeld) Batko as synonym of Brefeld’s E. radicans and
Zoophthora phytonomi (Arthur) Batko (syn. Entomophthora phytonomi Arthur).
Concurrently, he used the former species as a type for his genus and subgenus Zoophthora. In
France REMAUDIÈRE et al. /1976a/ studying Entomophthora isolates belonging to the
“sphaerosperma group” concluded that the fungus should be restricted to those isolates which
attack lepidopterous larvae, Diptera and other orders. And the Entomophthora isolates from
aphids the authors identified as morphologically similar Entomophthora phalloides Batko.
BEN-ZE’EV and UZIEL /1979/ using the same taxonomic principles as did REMAUDIÈRE et al.
/1976a/ and following Batko’s classification (with some restrictions) found that Z. radicans
attacking the yellow pecan aphid, Monellia costalis (Fitch), had the same spore dimensions
and morphology as E. sphaerosperma sensu stricto REMAUDIÈRE et al. /1976a/. Various
morphological, physiological and biochemical criteria were studied by GLARE et al. /1987/ to
reliably distinguish species Z. phalloides and Z. radicans. Despite the taxon separations
described above, the species is still considered to be a species complex /HUMBER 1983, 1990,
BAŁAZY 1993/. Recently, BAŁAZY /1993/ made thorough studies on morphology, biology and
particularly host specificity of genus Zoophthora what allowed him to distinguish and
describe 7 new species morphologically similar to Z. radicans.
Z. radicans is a very common and widespread species, as mentioned above it has a wide
host range including Lepidoptera, Diptera, Homoptera, Coleoptera, Orthoptera, Thysanoptera,
Hemiptera, and Hymenoptera /e.g. MILNER and SOPER 1981, SOPER and WARD 1981,
17
PAPIEROK et al. 1984, GLARE and MILNER 1991, KELLER 1991/. Although Z. radicans is
recorded from numerous insect groups, the literature suggests that individual isolates are
better adapted to infecting taxonomically related insect hosts /PAPIEROK et al. 1984, GOETTEL
et al. 1990/. For example, some strains were not able to infect species from orders other than
the order, from which they were isolated /PAPIEROK et al. 1984, MAGALHAES et al. 1988/.
However, some studies identified isolates with seemingly broader host ranges /POPRAWSKI et
al. 1992, FURLONG and PELL 1996/. According to Bałazy’s opinion /BAŁAZY 1993/, this
species is a specialized pathogen infecting caterpillars of Pieris spp., and application of this
name for pathogens of other insects is either incorrect or at least doubtful. Further, he
suggested that all earlier materials, excluding those from Pieris spp. caterpillars, should be
revised.
The fungus was recorded from many aphid species all over the world. It infected cereal
aphids /e.g. FENG et al. 1990, FENG and NOWIERSKI 1991/, aphids in potato fields /SHANDS et
al. 1972/, or pea aphid / PICKERING et al. 1989a/ in the USA. Several aphid species were
killed with the fungus in Israel /KENNETH and OLMER 1975, BEN-ZE’EV and UZIEL 1979/, in
France /THOIZON 1970/, in Switzerland /KELLER 1991/, and in Egypt /ABDEL-MALLEK et al.
2003/. It was also recorded from the black bean aphid in Sweden /GUSTAFSSON 1969/, and the
cabbage aphid in Yugoslavia /SIVČEV 1991, 1992/.
Z. radicans has a great potential for biological control. For example, isolates of this species
were released against the potato leafhopper, Empoasca fabae (Harris) in Illinois (the USA)
and epizootics occurred /MCGUIRE et al. 1987a,b/. The pathogen (an isolate from Israel) was
also released in Australia, where the fungus had never been recorded before, against the
spotted alfalfa aphid Therioaphis trifolii (Monell) f. maculata. The fungus established well in
the area and was able to survive as resting spores enabling it to persist from year to year and
subsequently spread on its own /MILNER and SOPER 1981, MILNER et al. 1982, MILNER and
MAHON 1985/.
Culture of this species can be established on Sabouraud’s agar supplemented with milk and
egg yolk /KELLER 1991, BAŁAZY 1993/.
10. Zoophthora canadensis (MacLeod, Tyrrell et Soper) Remaudière et Hennebert,

Mycotaxon 11: 301, 1980
Bas.: Entomophthora canadensis MacLeod, Tyrrell et Soper, Canad. J. Bot. 57: 2663-2672, 1979
Erynia canadensis (MacLeod, Tyrrell et Soper) Humber et Ben-Ze’ev, Mycotaxon 13: 509, 1981
Erynia subgenus Zoophthora canadensis (MacLeod, Tyrrell et Soper) Humber et Ben-Ze’ev ex
Ben-Ze’ev et Kenneth, Mycotaxon 14: 467, 1982b
The pathogen was found for the first time in populations of the woolly pine needle aphid,
Schizolachnus piniradiatae (Davidson) in Canada. During a study on the life history and
ecology of the aphid, a number of mycosed aphids were observed in the course of the summer
1960. The fungal pathogen was incorrectly identified as Empusa subgenus Entomophthora
aphidis Hoffman in Fresenius /GROBLER et al. 1962/. A continuing research into biology of
the aphid pathogen revealed the misidentification and a new fungal species, Entomophthora
canadensis MacLeod, Tyrrell et Soper was described /MACLEOD et al. 1979/. One year later,
REMAUDIÈRE and HENNEBERT /1980/ placed this taxon to the genus Zoophthora.
Z. canadensis is known only from populations of the aphid S. piniradiatae in red pine
plantations in Ontario, Canada / REMAUDIÈRE et al. 1979, REMAUDIÈRE 1993/.
Culture of the pathogen was established on coagulated hen’s egg yolk /MACLEOD et al.
1979/ and Grace’s insect tissue culture medium. However, growth on both media was
extremely slow with no conidia or resting spores produced /MACLEOD et al. 1979/.
18
11. Zoophthora occidentalis (Thaxter) Batko, Bull. Acad. Pol. Sci., Ser. Sci. Biol. 12: 404,
1964c
Bas.: Empusa subgenus Entomophthora occidentalis Thaxter, Mem. Boston Soc. Nat. Hist.4: 170-171,
1888
Erynia occidentalis (Thaxter) Humber et Ben-Ze’ev, Mycotaxon 13: 509, 1981
Erynia subgenus Zoophthora occidentalis (Thaxter) Humber et Ben-Ze’ev ex Ben-Ze’ev et
Kenneth, Mycotaxon 14: 467, 1982b
THAXTER /1888/ described a fungus infecting aphids on gray birch, Betula populifolia
Marsh, as Empusa subgenus Entomophthora occidentalis Thaxter. He recorded this pathogen
as frequently and commonly infecting the aphids in Maine (the USA). BATKO /1964c/
renamed this species as Zoophthora occidentalis (Thaxter) Batko and then placed it in the
subgenus Zoophthora BATKO /1966b/. Since further notes on the occurrence of this pathogen
were scarce, moreover biological and morphological characteristics of Z. phalloides closely
resembled those of Z. occidentalis, MIETKIEWSKI et al. /1981/ published a comprehensive
study on this taxon and made a comparison between conidial morphology and measurements
of both the fungi. BAŁAZY /1993/ presents his opinion that it is necessary to make a revision
of all the materials from aphids identified as Entomophthora sphaerosperma, Z. radicans, Z.
occidentalis and Z. phalloides, and to compare those morphologically similar species in order
to avoid any misidentifications.
The species has a distribution in the North and South America, Europe and Asia on
several, mostly, unidentified aphid species /BAŁAZY 1993/. Z. occidentalis was the most
frequent pathogen of the aphid M. persicae in Orono, Maine (the USA) in 1977 and it was
also collected from A. fabae on pigweed, Chenopodium album Linné. Morphologically
similar strain was also found in Poland from the aphid on Deschampsia ceaspitosa (Linné) P.
B. in the forest undergrowth /MIETKIEWSKI et al. 1981/. BATKO /1962/ listed this pathogen
among Polish entomopathogens from an aphid on nettle, Urtica dioica Linné, however, it was
found on few aphids only in one locality. Some individuals of aphids were killed by the
pathogen in Israel /BEN-ZE’EV et al. 1984/ and Chile /ARUTA and CARVILLO 1989/. E.
occidentalis was also reported from Anoecia sp. in France /THOIZON 1970/ and 3 individuals
of S. avenae were recorded to die from Z. occidentalis infection in the South-western Idaho
(the USA) /FENG et al. 1990/. BAŁAZY /1993/ states further three aphid species, Capitophorus
similis van der Goot, Impatientium asiaticum (Nevsky) and Sitobion fragariae (Walker), as
hosts for Z. occidentalis in Poland.
Strains of this fungus develop and sporulate well on artificial media containing egg yolk
/BAŁAZY 1993/.
12. Zoophthora orientalis Ben-Ze’ev et Kenneth, Phytoparasitica 9 (1): 33-42, 1981

Bas.: Erynia orientalis (Ben-Ze’ev et Kenneth) Humber, Ben-Ze’ev et Kenneth in Humber et Ben-
Ze’ev, Mycotaxon 13: 509, 1981
The pathogen is known only from the type locality in Israel where it was observed and
described from Aphis citricola van der Goot by BEN-ZE’EV and KENNETH /1981/.
13. Zoophthora anhuiensis (Li) Humber, Mycotaxon 34: 453, 1989

Bas.: Erynia anhuiensis Li, Acta Mycol. Sin. 5 (1): 1-6, 1986
This fungus was briefly described as Erynia anhuiensis from China where it was causing
epizootics in populations of the green peach aphid, M. persicae, in Anhui Province during late
19
autumn and early winter /LI 1986/. HUMBER /1989/ listed it among the species of the genus
Zoophthora in his sense. BAŁAZY /1993/ regards the taxon to be an intermediary form
(according to the original description) between Zoophthora and Furia as for primary conidia
and rhizoids as well as between Zoophthora and Erynia as for pseudocystidia. This species
has never been reported in other countries /FENG et al. 1998/.
Results of a bioassay on this fungus against M. persicae indicated that the pathogen could
be a promising agent for microbial control and competitive with other Zoophthora species
/FENG et al. 1998, XU et al. 2000/. Based on laboratory observations, the fungus is most
adaptive to cool climate or season with best effectiveness at 15°C /FENG and LI 2003/.
The fungus can be isolated and maintained on Sabouraud-milk-egg yolk medium /FENG et
al. 1998/.
Genus: Entomophthora Fresenius sensu Remaudière et Keller, Mycotaxon 11: 332, 1980
Syn.: Entomophthora Fresenius, Bot. Ztg. 14: 882, 1856 (nec Entomophthora Nowakowski, Pam.
Akad. Umiej., Wydz. Mat.-Przyr. 8: 175-176, 1883)
Empusa Cohn, Nova Acta Acad. Caes. Leop.-Carol. Germ. Nat. Curios. 25: 301-360, 1855 (nec
Empusa Lindley, Bot. Regn. sub. t.: 825, 1824)
Myiophyton Lebert, Neue Denkschr. Schweiz. Gesellsch. Naturwiss, 15: 26, 1856
Lamia Nowakowski, Pam. Akad. Umiej., Wydz. Mat.-Przyr. 8, 1883 (nec Lamia Endlicher, 1841)
Culicicola Nieuwland, Am. Midl. Nat. 4: 378, 1916
The generic name Entomophthora was in the past applied for practically all
entomophthoralean species except Massospora Peck. FRESENIUS /1856/ was the first who
used the name for the entomopathogenic fungi, instead of invalid Empusa (preoccupied by the
orchid Empusa Lindley) applied earlier by COHN /1855/. Nowakowski’s genus
Entomophthora /NOWAKOWSKI 1883/ was characterised by branched conidiophores, and
possession of both zygospores/azygospores and cystidia. THAXTER /1888/ suggested to
employ one genus, Empusa, for all insect attacking fungi. This genus comprised three
subgenera, from which Entomophthora included Nowakowski’s species of Entomophthora.
BATKO /1964e/ correctly pointed out that Entomophthora Fresenius is the later but valid
synonym of the invalid generic name Empusa Cohn, while Entomophthora Nowakowski or
Empusa (Entomophthora) Thaxter cannot be synonymized with the genus. Consequently he
restricted Entomophthora to three related species without rhizoids and with simple
conidiophores producing bell-shaped oligonucleate (< 10-12 nuclei) conidia. Finally,
REMAUDIÈRE and KELLER /1980/ emended the Batko’s definition of Entomophthora s. str.
(typified with Entomophthora muscae (Cohn) Fresenius) by removing the restriction as
regarding rhizoids and transferred some species of rejected Culicicola genus to the
Entomophthora s. str. (including E. planchoniana). This modification was later basically fully
accepted /BEN-ZE’EV and KENNETH 1982a, HUMBER 1989, BAŁAZY 1993/. KELLER /1987a/
listed 11 species within this genus. Later, BAŁAZY /1993/ added another two species. 4 new
species have been described since then and recently, KELLER /2002/ has reviewed the genus
and 5 new species have been described so that the total number of Entomophthora species has
increased to 22.
14. Entomophthora planchoniana Cornu, Bull. Soc. Bot. France 20: 189, 1873
Bas.: Myiophyton planchonianum (Cornu) Arx, The genera of fungi: 51, 1970
(nec Empusa planchoniana (Cornu)? ex Thaxter, Mem. Boston Soc. Nat. Hist. 4: 165-166, 1888)
E. planchoniana was first observed and described in southern France as a natural enemy of
aphids on elderberry /CORNU 1873/. In point of fact, E. planchoniana is probably the first
20
entomophthoralean fungus described from aphid host, at all. Though CORNU /1873/ gave no
additional data on the fungus, at present it is considered one of the most important fungal
pathogen of aphids in Europe. It is affecting many aphid species in dry and moderately humid
habitats /BAŁAZY 1993/. It does not occur in dense, humid crops and seems to prefer dry
habitats like tall plants, bushes, and trees often causing epizootics /KELLER 1987a/. In Poland
the species was found on 15 different aphid species in plenty of localities all over the country
during summer and autumn /BAŁAZY 1993/. In Switzerland the fungus infected 16 species of
aphids /KELLER 1987a/ and during a survey of aphid pathogens in the South Africa the fungus
infected 6 different aphid species /HATTING et al. 1999a/.
Generally, the fungus is distributed all over the world but primarily in Europe where it
causes epizootics within aphid populations /HUMBER and FENG 1991/. E. planchoniana has
been reported as important natural enemy of cereal aphids in Europe /e.g. DEAN and WILDING
1971, KENNETH and OLMERT 1975, DEDRYVER 1981, 1983, COREMANS-PELSENEER et al.
1983, PAPIEROK and HAVUKKALA 1986, OZINO et al. 1988, STEENBERG and EILENBERG 1995,
ŠTALMACHOVÁ and CAGÁŇ 2000, EILENBERG 2002/, in the South and North Africa /HATTING
et al. 1999a, 2000, ABDEL-MALLEK et al. 2003/ as well as in the USA /FENG et al. 1991/. The
fungus is also parasiting pea aphid /WILDING 1975, PICKERING and GUTIERREZ, 1991/, A.
kondoi in Australia /MILNER et al. 1980/, Phorodon humuli (Schrank) /KREJZOVÁ 1979/,
several aphids in potato fields in the USA /SHANDS et al. 1972/, A. fabae /GUSTAFSSON 1969,
WILDING and PERRY 1980/ as well as B. brassicae /SIVČEV 1991, 1992/. The fungus was also
recorded from “non-agricultural aphids”, Drepanosiphum acerinum (Walker) in Switzerland
/KELLER 1987b/ or Elatobium abietinum Walker in Iceland /AUSTARÅ 1997, NIELSEN et al.
2001b/ as well as different aphid species in Denmark /EILENBERG 2002/.
For many years E. planchoniana was reported not to grow in vitro, what was the main
obstacle for its use in the biological control. There was only HOLDOM /1983/ who succeeded
in isolation of strains morphologically similar to E. planchoniana (the author stated the aphid
pathogenic species as E. planchoniana). The strains were isolated in a liquid medium based
on foetal calf serum, glucose and neopeptone. After a transfer of the isolate to a solid medium
(SEMA) a sporulation occurred. However, the identification of the isolates was corrected later
to Entomophtora chromaphidis Burger and Swain /HUMBER and FENG 1991/. Recently,
FREIMOSER et al. /2001/ have been successful in isolation and in vitro cultivation of the aphid
pathogenic fungus for the first time. The strains were isolated from Ovatus crataegarius
(Hem.) and cultivated on Grace’s insect tissue cell culture medium supplemented with
additional nutriments. Although the cultures did not sporulate, it raised hopes for perspective
exploitation of the fungus in the biological control of aphids.
15. Entomophthora chromaphidis Burger et Swain, J. Econ. Entomol. 11: 288, 1918
Bas.: Culicicola chromaphidis (Burger et Swain) Batko, Bull. Acad. Pol. Sci., Ser. Sci. Biol. 12: 404,
1964c
This is the next Entomophthora species infecting aphids. The pathogen was reported as
significant mortality factor of walnut aphid, Chromaphis juglandicola (Kaltenbach) during a
period of exceptionally hot weather in southern California /BURGER and SWAIN 1918/. For
many years E. chromaphidis was considered being a synonym for E. planchoniana
/GUSTAFSSON 1965, MACLEOD et al. 1976, WATERHOUSE and BRADY 1982/ until the two
species were separated and justified /HUMBER and FENG 1991/. E. chromaphidis occurs in the
North America and Australia and is characterized by having smaller primary conidia and
larger nuclei, whereas E. planchoniana is more widely distributed (with primarily European
distribution) and has bigger primary conidia and smaller nuclei. E. chromaphidis has been
successfully isolated and cultivated /HOLDOM 1983, HUMBER and FENG 1991/. In spite of
21
aforementioned differences BAŁAZY /1993/ prefers not to separate those taxons and preserved
them under the name E. planchoniana. Recently, genetic analysis could not separate it from
E. planchoniana /JENSEN and EILENBERG 2001/. KELLER /2002/ considers E. chromaphidis a
valid species.
In addition to the type host, Ch. juglandicola, the fungus was identified also from M.
dirhodum, S. avenae, Schizaphis graminum (Rondani), and Diuraphis noxia (Mordvilko)
/FENG at al. 1990, 1991a,b, FENG and NOWIERSKI 1992a, WRAIGHT et al. 1993/ and M.
persicae /KISH et al. 1994/ in the USA and from A. kondoi in Australia /HOLDOM, 1983/. This
taxon has newer been reported from Europe.
Genus: Entomophaga Batko emend. Humber, Mycotaxon 34: 447-448, 1989

Syn.: Entomophaga Batko, Bull. Acad. Pol. Sci., Ser. Sci. Biol. 12: 325-326, 1964b
This genus, based on Entomophthora grylli Fresenius, was proposed by BATKO /1964b/ to
include species with multinucleate spores and simple conidiophores but without rhizoids.
BATKO /1974/ tried to distinguish Conidiobolus and Entomophaga species on basis of trophic
relations, saprophytism or facultative zoopathogenicity in the first versus arthropod parasitism
in the second group. However, no sufficient criteria were provided to reliably distinguish
species placed in genera Entomophaga, Culicicola Nieuwland or morphologically similar
genus Conidiobolus Brefeld. The lack of these criteria seemingly redounded to transfer all the
species of Entomophaga and some species of the Culicicola to genus Conidiobolus by
REMAUDIÈRE and KELLER /1980/. New criterion based on nuclear morphology of the fungi
propounded by HUMBER /1981/ did provide suitable criterion to differentiate Conidiobolus
species with ancylistoid nuclei from those of Entomophaga with easily staining
entomophthoroid nuclei. HUMBER /1989/ listed 10 species under this generic name. BAŁAZY
/1993/ recognizes 14 species of the genus in his monograph (with 2 new species) and recently
two Eryniopsis species were transferred to the genus /Hajek et al. 2003/. There is only one
species that exhibits aphidophagous properties.
16. Entomophaga pyriformis (Thoizon) Bałazy, Flora of Poland. Fungi (Mycota) 24: 119,
1993
Bas.: Entomophthora pyriformis Thoizon, Entomophaga 12: 303-307, 1967
Among fungus infected aphids collected in different parts of France, THOIZON /1967/
found specimens of Rhopalosiphum insertum (Walker) at La Verne being killed by species
with pyriform conidia. The author described and named the fungus Entomophthora pyriformis
Thoizon. Under laboratory conditions the pathogen also infected Rhopalosiphum padi
(Linné), Sitobion avenae (Fabricius) and A. fabae. THOIZON /1970/ recorded this fungus from
Forda formicaria von Heyden, Tetraneura ulmi (Linné), Linosiphon galii Mamont., and R.
insertum. This aphidophagous species is known only from the type locality and has not been
observed by other mycologists in aphid colonies. The taxon was not mentioned in several
comprehensive taxonomic works /WATERHOUSE and BRADY 1982, HUMBER 1989, BEN-
ZE’EV and KENNETH 1982a/ until that of Bałazy’s one /BAŁAZY 1993/. Bałazy allocated this
species as comb. nov. in the genus Entomophaga.
E. pyriformis grows quite readily and may be cultured on Sabouraud dextrose agar and on
egg yolk with glucose /THOIZON 1967/.
Genus: Batkoa Humber, Mycotaxon 34: 446-447, 1989
22
The genus was established and typified with Batkoa apiculata (Thaxter) Humber by
HUMBER /1989/ to distinguish certain species from the genus Entomophaga on the ground of
marked differences in the structure of conidiophores and with the presence of rhizoids as
supporting character. KELLER /1987a, 1991/ rejected the subdivision of the genus
Entomophaga into Entomophaga and Batkoa as proposed by Humber, since he considered the
insufficiently researched criteria for the separation premature and all the Humber’s species
transferred to Entomophaga Batko. Several years later KELLER /1999/, however, accepted the
genus Batkoa Humber with Humber’s species. The genus consists of five obligately
entomopathogenic species /HUMBER 1989, BAŁAZY 1993, KELLER 1999/. Though all the five
species differ morphologically from Entomophaga the current results of molecular analyses
demonstrate that Batkoa is closely related to Entomophaga /HAJEK et al. 2003/. Two species
have been recorded from aphids.
17. Batkoa apiculata (Thaxter) Humber, Mycotaxon 34: 446, 1989

Bas.: Empusa apiculata Thaxter, Mem. Boston. Soc. Nat. Hist. 4: 163-164, 1888
Lamia apiculata (Thaxter) Lakon, Z. angew. Entomol. 5: 173-174, 1919
Culicicola apiculata (Thaxter) Batko, Bull. Acad. Pol. Sci., Ser. Sci. Biol. 12: 404, 1964d
Entomophthora apiculata (Thaxter) Gustafsson, Lantbrukshogsk. Ann. 31: 131-133, 1965
Conidiobolus apiculatus (Thaxter) Remaudière et Keller, Mycotaxon 11: 330, 1980 (nec
Conidiobolus apiculatus (Thaxter) Remaudière et Keller in Keller, Sydowia 40: 128-130, 1987a)
Syn.: Entomophaga domestica Keller, Sydowia 40: 141-143, 1987a
This species was found in various insect species collected in Maine and North Carolina,
the USA. THAXTER /1888/ suggested a provisional name Empusa apiculata for the fungus
until more complete description of three other species, to which the taxon was closely allied,
were reported. LAKON /1919/ transferred the species to genus Lamia. Lamia Nowakowski,
however, was a later homonym of Lamia Endl. Therefore, Culicicola Nieuwland is in opinion
of BATKO /1964d/ the valid name of this genus. In Sweden, GUSTAFSSON /1965/ found an
entomophagous species, identified as Entomophthora apiculata, on a number of Diptera and
Psocoptera species. However, conidia were smaller than those of the Thaxter’s strains and, in
addition, microconidia were observed. MACLEOD and MÜLLER-KÖGLER /1973/ believed that
the Swedish strains are hardly allied to E. apiculata and they gave them to relation with
Conidiobolus pseudococci (Speare) Tyrrell et MacLeod and Conidiobolus coronatus (Cost.)
Tyrrell et MacLeod. REMAUDIÈRE and KELLER /1980/ revised the genus Conidiobolus and
placed the species to this genus as Conidiobolus apiculatus. KELLER /1987a/ misidentified a
pathogen of Diptera and larvae of Tenthredinidae (Hymenoptera) in Switzerland as C.
apiculatus, species with typical ancylistoid nuclei proving its affinity with genus
Conidiobolus (family: Ancylistaceae). However, HUMBER /1989/ re-examined Thaxter’s
collection and stated that all materials identified by Thaxter as Empusa apiculata, which had
been considered a basionym for C. apiculatus, had entomophthoroid nuclei. Consequently, he
transferred the Thaxter’s species to the family Entomophthoraceae as a type of the new genus
Batkoa and suggested to describe the Keller’s fungus as a new Conidiobolus species. Further,
a new taxon, Entomophaga domestica Keller, described by KELLER /1987a/ in Switzerland as
pathogen of several Diptera and Coleoptera species in glasshouse was treated as synonym for
Batkoa apiculata /HUMBER 1989/.
B. apiculata is polyphagous species. It has been reported from all continents as pathogen of
adult insects of different groups /BAŁAZY 1993/. THOIZON /1970/ reported a species
Entomophthora apiculata from several aphid species in various parts of France, as well.
Altogether three aphid species were collected being infected with this pathogen:
23
Macrosiphoniella mutellinae Börner, Sitobion avenae (Fresenius) and Rhopalosiphum padi

Linné. It is also the only record of the pathogen from aphids.
The fungus develops well and sporulates on artificial media, especially those containing
egg yolk and Sabouraud’s dextrose agar /GUSTAFSSON 1965, KELLER 1987a, BAŁAZY 1993/.
18. Batkoa major (Thaxter) Humber, Mycotaxon 34: 446, 1989

Syn.: Empusa apiculata var. major Thaxter, Mem. Boston. Soc. Nat. Hist. 4: 164-165, 1888
Lamia apiculata var. major (Thaxter) Lakon, Z. angew. Entomol. 5: 174, 1919
Bas.: Entomophthora major (Thaxter) Gustafsson, Lantburtkshogsk. Ann. 31: 133-134, 1965
Conidiobolus major (Thaxter) Remaudière et Keller, Mycotaxon 11: 331, 1980 (nec Conidiobolus
major (Thaxter) Remaudière et Keller in Keller, Sydowia 40: 132-134, 1987a)
This fungus, found on naturally infected adults of Ptilodactyla serricollis (Say)

(Coleoptera: Ptilodactylidae) collected in North Carolina, the USA, was named and described
by THAXTER /1888/ as Empusa apiculata var. major, a variant of the species E. apiculata.
GUSTAFSSON /1965/ found this pathogen infecting aphid Myzodium modestum (Hottes), but he
distinguished it from the species E. apiculata due to clear differences in size and appearance
of conidia and the size of resting spores. He suggested a new name Entomophthora major as a
distinct species and not a variant of E. apiculata. BATKO /1964c,d/ placed this fungus in the
genus Culicicola because of presence of rhizoids and multinucleate spores borne on simple
conidiophores. KING /1976a,b/, according to his comprehensive study on genus Conidiobolus,
believed that the species belonged to this genus. This conclusion was affirmed by
REMAUDIÈRE and KELLER /1980/, as well. HUMBER /1989/, after investigation of Thaxter’s
authentic collections of E. apiculata var. major, placed this species in his new genus Batkoa
(family Entomophthoraceae) on the basis of entomophthoroid nuclei of the taxon and other
morphological features. He also stated that E. apiculata var. major is basionym for
Conidiobolus major (Thaxter) Remaudière et Keller. KELLER /1987a/ misidentified a
pathogen of spittlebugs (Homoptera: Cercopidae) in Switzerland as C. major, species with
typical ancylistoid nuclei proving its affinity with genus Conidiobolus (family:
Ancylistaceae). HUMBER /1989/ suggested a need to describe the Keller’s species with
features of C. major as a new Conidiobolus species and Humber simultaneously synonymized
Keller’s new species Entomophaga limoniae Keller, entomopathogen of Diptera (Limoniidae)
in Switzerland, with B. major. KELLER /1991/ in view of Humber’s taxonomic corrections
described new Conidiobolus species, but persisted in his opinion that E. limoniae is a separate
species.
This species is reported from plenty of localities in the North and South America, Europe
and Asia as a pathogen on several insect species of different orders /BAŁAZY 1993/. The
fungus was only recorded from aphid in Sweden by GUSTAFSSON /1965/.
The fungus grows and develops well on media containing egg yolk and Sabouraud’s
dextrose agar /GUSTAFSSON 1965, BAŁAZY 1993/.
Family: Neozygitaceae Ben-Ze’ev et Kenneth in Ben-Ze’ev et al., Mycotaxon 28: 313-326,

1987
Genus: Neozygites Witlaczil, Arch. F. Mikroskop. Anat. 24: 599-603, 1885
Syn.: Neozygites Remaudière et Keller, Mycotaxon 11: 331-332, 1980
Empusa subgenus Triplosporium Thaxter, Mem. Boston Soc. Nat. Hist. 4: 152, 1888
Triplosporium (Thaxter) Batko, Bull. Acad. Pol. Sci., Ser. Sci Biol. 12: 324-325, 1964b
Thaxterosporium Ben-Ze’ev et Kenneth in Ben-Ze’ev et al., Mycotaxon 28: 323, 1987
24
The genus comprises a relatively homogenous group of fungi. The nuclear structure and
the nuclear behaviour during mitosis of these fungi differ from the other entomophthoralean
species /BUTT and HEATH 1988, BUTT and HUMBER 1989/. Therefore the genus was placed in
a distinct family Neozygitaceae proposed and described by BEN-ZE’EV et al. /1987/ what was
also maintained by HUMBER /1989/ and KELLER /1991, 1997/. Recently, this family has not
been distinguished by BAŁAZY /1993/ and he left all the Neozygites species organized within
the family Entomophthoraceae.
The genus was originally established by WITLACZIL /1885/. THAXTER /1888/, however,
proposed the subgenus Triplosporium (within the genus Empusa) for species characterised by
smoky conidia and black, oval zygospores. BATKO /1964b/ elevated the Thaxter’s subgenus to
the generic rank and listed three species in the genus according to THAXTER /1888/. Based on
nomenclatural priority, REMAUDIÈRE and KELLER /1980/ placed Triplosporium in synonymy
under Neozygites. HUMBER et al. /1981/ proposed to conserve the name Triplosporium against
Neozygites, but the proposal failed. BEN-ZE’EV and KENNETH /1982a/ preferred the Batko’s
generic name Triplosporium after its emendation. At present a validity of the generic name
Neozygites has been accepted by most taxonomic authorities /HUMBER 1989, KELLER 1991,
1997, BAŁAZY 1993/.
Neozygites consists of a homogenous group of 15 different fungi attacking small pterygote
insects (Homoptera, Thysanoptera) and mites /KELLER 1997/. Recently, however, a
Neozygites species was also discovered attacking the apterygote insect, springtail Sminthurus
viridis Linné /STEENBERG et al. 1996, DROMPH et al. 2001/ and one new species was
described /KELLER and STEENBERG 1997/. Species of this genus can be divided into groups on
the basis of their morphology and life cycle. All the aphidophagous species belong to the
Neozygites fresenii-group, except for Neozygites lecanii, which seems to be closer related to
this group as well, but deficiencies in its description make the obstacle for a definite decision
/KELLER 1997/.
Out of the arthropod-pathogenic genera of Entomophthorales the fungi of Neozygites genus
are the best adapted to tropical conditions /KELLER 1997/. Five of the group of 15 species are
found in the tropics and three of them are exclusively found in the regions. The genus
includes 6 aphid-pathogenic species.
19. Neozygites fresenii (Nowakowski) Remaudière et Keller, Mycotaxon 11: 332, 1980
Bas.: Empusa freseniana Nowakowski, Bot. Zeitung (Berlin) 40: 561, 1882, (nomen nudum)
Empusa fresenii Nowakowski, Pam. Akad. Umiej. Kraków 8: 171-172, 1883
Empusa subgenus Triplosporium fresenii Nowakowski ex Thaxter, Mem. Boston. Soc. Nat. Hist. 4:
167-169, 1888
Entomophthora fresenii (Nowakowski) Gustafsson, Lantburkshogsk. Ann. 31: 141-142, 1965
Triplosporium fresenii (Nowakowski) Batko, Bull. Acad. Pol. Sci., Ser. Sci. Biol. 12: 325, 1964b
Syn.: Neozygites aphidis Witlaczil, Arch. Mikr. Ant. 24: 599-603, 1885
Entomophthora neri Gustafsson, Lantburkshogsk. Ann. 35: 243, 1969
This species named and described as Empusa fresenii by NOWAKOWSKI /1883/ was found
on naturally infected aphids collected in Poland. This is the first described member of the
Neozygites genus. Later, WITLACZIL /1885/ described an aphid infection observed in Aphis
(Hyalopterus) arundinis Fabricius as Neozygites aphidis, believing the pathogen to be a
gregarine protozoan. THAXTER /1888/, unaware of the description by Witlaczil, placed E.
fresenii in a new subgenus, Triplosporium. GIARD /1888/ was the first who noted that
Witlaczil had in fact described resting-spore stage of E. fresenii and that Triplosporium and
Neozygites were synonymous. RUDRAIAH and USMAN /1955/ reported Entomophthora sp.
from the oleander aphid, Aphis nerii Fonscolombe, in India. Gustafsson in a subsequent
25
reference to this fungus /GUSTAFSSON 1969/ referred to it as a new species Entomophthora

neri Gustafsson. MACLEOD and MÜLLER-KÖGLER /1973/ suggested that E. neri is not a new
species, but the resting-spore stage of N. fresenii. Until 1980, when REMAUDIÈRE and KELLER
/1980/ replaced Triplosporium (Thaxter) Batko with the genus Neozygites Witlaczil, this
fungus was mentioned in the literature as Triplosporium fresenii.
N. fresenii is a widely occurring fungal pathogen of aphids and has been recorded in
various countries. It has been reported from Europe /e.g. THOIZON 1970, DEDRYVER 1978,
KELLER 1991, SIVČEV 1991, 1992, BAŁAZY 1993, STEENBERG and EILENBERG 1995/, the
North America /e.g. SOPER and MACLEOD 1963, FENG et al. 1990/, Israel /BITTON et al.
1979/, the South Africa /HATTING et al. 1999a/, South Pacific /KELLER 1997/ and Australia
/MILNER and HOLDOM 1986/. Aphids of the genus Aphis, especially species Aphis fabae /e.g.
GUSTAFSSON 1965, ROBERT et al. 1973, DEDRYVER 1978/ and Aphis gossypii Glover /e.g.
SILVIE and PAPIEROK 1991, STEINKRAUS et al. 1991, 1995/ are considered the major host
organisms for the pathogen. N. fresenii causes widespread epizootics in the aphid populations
over the world. Moreover, it is best adapted to hot, humid, even tropical conditions
/REMAUDIÈRE 1977, STEINKRAUS et al. 1991, KELLER 1997/, but the epizootics has already
been reported in Iceland, as well /AUSTARÅ 1997, NIELSEN 2001/. Epizootiology of the
fungus in annual cropping ecosystems has been well studied /STEINKRAUS et al. 1991, 1993,
1996b, 1999/, but its commercial exploitation is limited with absence of any isolates.
Despite numerous attempts to grow N. fresenii on artificial media, it has not been isolated
/KELLER 1997/.
20. Neozygites microlophii Keller, Sydowia 43: 82, 1991

N. microlophii was common fungus and caused epizootics among dense populations of the
nettle aphid species, Microlophium carnosum Buckton, in Switzerland where it was
discovered for the first time and described by KELLER /1991/. The fungus could be found
within the aphid populations between mid-June and mid-July. KELLER /1997/ thinks that some
collections identified as morphologically similar N. fresenii in the past might belong to this
species, especially those from larger aphids.
Resting spores of the pathogen were later identified from the nettle aphid in Poland, as well
/BAŁAZY 1993/.
The species has only been known from the type host.
21. Neozygites turbinata (Kenneth) Remaudière et Keller, Mycotaxon 11: 322, 1980
Bas.: Entomophthora turbinata Kenneth, Mycotaxon 6: 381-390, 1977
Thaxterosporium turbinatum (Kenneth) Ben-Ze’ev et Kenneth in Ben-Ze’ev, Kenneth et Uziel,
Mycotaxon 28: 313-326, 1987
The pathogen was discovered in Israel on aphid Pterochloroides persicae (Cholodk.) in

1977 and named Entomophthora turbinata Kenneth /KENNETH 1977/. The species was closely
related to the genus Triplosporium (Thaxter) Batko, but no secondary conidia were observed
and both hyphal bodies and primary spores contained 7-11 nuclei. However, Triplosporium
was described by BATKO /1964b/ to have tetranucleate spores. REMAUDIÈRE and KELLER
/1980/ listed this fungus under the generic name Neozygites, whereas BEN-ZE’EV et al. /1987/
separated it as a representative of the new genus, Thaxterosporium Ben-Ze’ev et Kenneth.
KELLER /1991/, who revealed another species with number of nuclei in spores and hyphal
bodies other than four, did not see a reason for a separate genus for N. turbinata and
synonymized the genus Thaxterosporium with Neozygites.
26
In Switzerland the species was observed in colonies of aphid Tuberolachnus salignus

Gmelin on Salix sp. from the end of August till the beginning of November, where it caused
strong epizootics /KELLER 1991, 1997/.
At present, the fungus is known from the abovementioned aphid species in Israel and
Europe /BAŁAZY 1993, KELLER 1997/.
22. Neozygites lageniformis (Thaxter) Remaudière et Keller, Mycotaxon 11: 322, 1980
Bas.: Empusa subgenus Triplosporium lageniformis Thaxter, Mem. Boston. Soc. Nat. Hist. 4: 169,
1888
Triplosporium lageniformis (Thaxter) Batko, Bull. Acad. Pol. Sci., Ser. Sci. Biol. 12: 403, 1964b
Entomophthora lageniformis (Thaxter) MacLeod et Müller-Kögler, Mycologia 65: 858, 1973
The species is incompletely described. THAXTER /1888/ described this fungus as Empusa
(Triplosporium) lageniformis Thaxter for the first time in the USA from unidentified aphid
species. It was suggested that N. lageniformis frequently occurred in association with Z.
occidentalis. The species is known from the USA and Chile on aphids Myzocallis coryli
(Goetze) on Corylus avellana Linné and unidentified aphid species on Betula populifolia
Marsh and Solidago sp. /ARUTA and CARVILLO 1989, BAŁAZY 1993/.
23. Neozygites lecanii (Zimmermann) Bałazy, Flora of Poland, Fungi (Mycota) 24: 139-
140, 1993
Bas.: Empusa lecanii Zimmermann, Meded. Lands Plantent. 44: 25-27, 1901
Triplosporium lecanii (Zimmermann) Ben-Ze’ev et Kenneth, Mycotaxon 14: 436, 1982a
Derexia lecanii Narasimhan, Indian Phytopathol. 23: 16, 1970 (nomen confusum)
Derexiomyces lecanii Narasimhan et Thirumalachar, Abstr. 2. Intern. Mycol. Congres Tampa: 466,
1977 (nomen confusum)
The species was for the first time observed on Lecanium viridae Green (Homoptera:
Coccidae) in Java in 1901 and described as Empusa lecanii /ZIMMERMMAN 1901/. The
species is incompletely described and known only from South East Asia /BAŁAZY 1993,
KELLER 1997/. HUMBER /1990/ listed this species among fungi parasiting aphids.
24. Neozygites cinarae Keller, Sydowia 49 (2): 137-138, 1997

The species was collected in a plantation of Picea abies Karst. within colonies of aphid,
Cinara pilicornis (Hartig) (the type host) in Switzerland between the end of June and the
beginning of July in 1991 and 1995 and described by KELLER /1997/. N. cinarae is
morphologically and taxonomically closely related to N. fresenii and N. microlophii. It can be
distinguished mainly by larger conidia, number of nuclei in conidia, and length of capillary
tube.
The pathogen has been only known from the type host and the type locality.
Family: Basidiobolaceae Claussen in Engler et Gilg, Syll. Pflanzenfam.: 45, 1924

Genus: Basidiobolus Eidam, Beitr. Biol. Pflanz. 4: 183-251, 1886
The genus includes 5 species /BEN-ZE’EV and KENNETH 1982a, HUMBER 1989, BAŁAZY
1993/ and most of them are saprobes in soil or plant detritus, but they may also occur as
colonisers of the guts of amphibian and reptiles or as pathogens of vertebrate (including
humans) /COREMANS-PELSENEER 1974/. Members of the genus are common mostly in
subtropical and tropical regions /BAŁAZY 1993/. The evidence for infection of insects by
Basidiobolus is uncertain even though there have been some records of Basidiobolus from
27
insects as occasional pathogen /KING and HUMBER 1981, BAŁAZY 1993/ or some insect
species have been successfully inoculated in the laboratory /KREJZOVÁ 1972b, EGINA and
SHAGUNINA 1977, ČUDARE 1982/. On the other hand some authors state that most attempts to
show that Basidiobolus invades insects failed /KING and HUMBER 1981/.
Latest phylogenetic analyses of the Entomophthorales based on ribosomal DNA suggest
that the genus Basidiobolus may not belong to the Entomophthorales /JENSEN et al. 1998/.
25. Basidiobolus ranarum Eidam, Beitr. Biol. Pflanz. 4: 194-239, 1886

A description of this taxon does not differ from the genus characteristics. The species is
distributed in all continents in amphibian and reptile excrements, soil and organic detritus. In
one case the fungus was isolated from horse leg scratch. In Poland strains of the species have
been isolated from excrements of Bufo bufo Linné and as contaminants of entomophthoralean
cultures from insects collected in moist habitats /BAŁAZY 1993/.
Basidiobolus species are usually omitted from reviews of arthropod-pathogenic fungi as
they are primarily considered a soil saprobes /ZIMMERMANN 1978, LATGÉ and PAPIEROK
1988, HUMBER 1990, 1997, KELLER 1991, 1999/. However, B. ranarum have already been
successfully isolated from insects (including aphids), as well. One strain of B. ranarum was
obtained from R. padi in France in 1970 /LATGÉ 1975/. But a presence of the species in fields
is accidental /REMAUDIÈRE et al. 1976b/. Basidiobolus sp. was collected from cereal aphids in
the North Africa /ABDEL-MALLEK et al. 2003/. Basidiobolus sp. was successfully isolated
from M. persicae in Russia /EGINA et al. 1977/, reared on colonies of M. persicae, and
consequently tested against aphids and mites /EGINA and SHAGUNINA 1977/. In laboratory
bioassay S. avenae received infection of Basidiobolus sp. after 18 hours contact with soil
samples collected in the field /NIELSEN et al. 1998/.
Development in artificial cultures is variable, depending on a strain and a composition of
media. Generally, isolates can be easily obtained on Sabouraud’s dextrose agar /BAŁAZY
1993/.
Family: Ancylistaceae v. Thieghem, Traité de Botanique: 1007, 1884

Genus: Conidiobolus Brefeld emended Humber, Mycotaxon 34:455-456, 1989
The genus was established and typified with Conidiobolus utriculosus Brefeld by Brefeld
in 1884 /KING and HUMBER 1981/. The criterion used to delimit this fungal genus from
Entomophthora s.l. was predominantly saprophytism of the Conidiobolus species versus
parasitism of the latter /BEN-ZE’EV and KENNETH 1982a/. Some insect parasitic species were,
however, later transferred from Entomophthora s.l. to Conidiobolus /BATKO 1964a,
SRINIVASAN and THIRUMALACHAR 1964, TYRRELL and MACLEOD 1972/. KING /1977/ also
noted a morphological similarity of several entomopathogenic Entomophthora s.l. species
with Conidiobolus. Extending the King’s concept of Conidiobolus, REMAUDIÈRE and KELLER
/1980/ proposed to include all the entomophthoralean forms with spherical, ovoid and
pyriform, unitunicate and multinucleate conidia to the genus Conidiobolus. With a result that
also almost all the species of Entomophaga Batko emended Humber and Batkoa Humber
were listed under this name. HUMBER /1989/ emended description of Conidiobolus Brefeld,
providing clear morphological and developmental separation of this genus from Entomophaga
and Batkoa. BEN-ZE’EV and KENNETH /1982a/ proposed to divide this genus into three
subgenera (Delacroixia, Conidiobolus and Capillidium) on the basis of peculiar forms of
secondary conidial sporulation. BAŁAZY /1993/ accepted this arrangement.
Most of Conidiobolus species are saprobic in detritus, but some are also known from insect
and vertebrate mycoses /BAŁAZY 1993/. They are often isolated from soil samples /e.g.
COREMANS-PELSENEER et al. 1983, ALI-SHTAYEH et al. 2002, Keller et al. 2003/.
28
Among the 27 species recognised by KING /1977/ only two were not originally described
as species of Conidiobolus. BEN-ZE’EV and KENNETH /1982a/ added further 4 species
including three entomopathogenic species recorded earlier under the generic names Empusa
or Entomophthora s.l. After detailed studies of karyological characteristics, two from among
them (Batkoa apiculata and Batkoa major) were included into the family of
Entomophthoraceae, whereas Entomophthora destruens Batko et Weiser was transferred to
Conidiobolus /BEN-ZE’EV 1982/. Subsequently four new species were described /BAŁAZY et
al. 1987, KELLER 1991, BAŁAZY 1993/, so the actual number of the species is 34. Out of 9
parasitic Conidiobolus species five have been recorded from aphids.
26. Conidiobolus coronatus (Costantin) Batko, Entomophaga, Mem. Hors., Ser. 2: 129,
1964a
Bas.: Boudierella coronata Costantin, Bull. Soc. Mycol. Fr. 13: 40, 1897
Delacroixia coronata (Costantin) Sacc. et Sydow, Syll. Fung. 14: 457, 1899
Entomophthora coronata (Costantin) Kevorkian, J. Agaric. Puerto Rico 21: 198, 1937
Conidiobolus coronatus (Costantin) Srinivasan et Thirumalachar, Mycopathol. Mycol.Appl. 24:
294-296, 1964
Conidiobolus subgenus Delacroixia coronatus (Costantin) Tyrrell et MacLeod, J. Invertebr. Pathol.
20: 12, 1972
Conidiobolus subgenus Delacroixia coronatus (Costantin) Batko in Bałazy, Flora of Poland. Fungi
(Mycota) 24: 78-79, 1993
Syn.: Conidiobolus villosus Martin, Bot. Gaz. 80: 311-318, 1925
This fungus, isolated more than 100 years ago from a culture of Agaricus campestris Fr.
(or from a dead insect between the lamellae), was named and described by Costantin in 1897
as Boudierella coronata Costantin /MACLEOD and MÜLLER-KÖGLER 1973/. Since then it has
been re-isolated from numerous diverse sources /HALL and DUNN 1957, GUSTAFSSON 1965,
MACLEOD and MÜLLER-KÖGLER 1973, KING 1979, KELLER 1987a, FENG et al. 1990/. It has
also been treated under seven different names and as belonging to four different genera. C.
coronatus is one of two Conidiobolus species listed by KING /1977/, which was not originally
described as a species of Conidiobolus. KEVORKIAN /1937/ transferred this species to
Entomophthora genus mainly because it also attacked insects. It was BATKO /1964a/ who
correctly offered to transfer Entomophthora coronata to the genus Conidiobolus. BEN-ZE’EV
and KENNETH /1982a/ and BAŁAZY /1993/ placed this pathogen to the subgenus Delacroixia
based largely on the ability to produce microconidia from the primary or secondary conidium.
At present the species is considered a species complex and the rather great variability or
intraspecific diversity requires detailed study /BATKO 1974, HUMBER 1983, 1990/.
The fungus is distributed all over the world. It is recorded as a widespread soil saprophyte
utilising a variety of substrates, including detritus, living plants, different dead arthropods,
and occasionally mammals (including humans) /MACLEOD and MÜLLER-KÖGLER 1973, KING
1979, KELLER 1987a, BAŁAZY 1993/. Nevertheless the species is known to cause diseases in
insects, as well. Aside from other insect orders, it was identified from several aphid species in
Poland /e.g. BATKO 1962, 1966a, BAŁAZY et al. 1990, BAŁAZY 1993/ and France /THOIZON
1970, PAPIEROK 1985/, from the cabbage aphid in Yugoslavia /SIVČEV 1991/, Elatobium
abietinum in Wales /AUSTARÅ 1997/, from the cereal aphids in the South and North Africa
/HATTING et al. 1999a, ABDEL-MALLEK et al. 2003/, or in the USA /FENG et al. 1990/, from
the green peach aphid in Israel /GINDIN and BEN-ZE’EV 1994/, or from Macrosiphum
euphorbiae (Thomas) in the USA /SHANDS et al. 1972/. Strains of various origin, saprobes
and pathogens, proved to be pathogenic for aphids, however, their virulence showed obvious
differences /PAPIEROK 1985, PAPIEROK et al. 1993/. It was demonstrated that C. coronatus
should be regarded as a non-primary pathogen under natural conditions. It invades secondarily
29
insects, in which death resulted from infection by a parasitic Entomophthorales or from

another cause. In laboratory, saprophytic species of Conidiobolus were as infective for aphids
as were species active under natural conditions, but were more rapid in the action, probably
due to a production of toxins /PAPIEROK 1986/. Recently, a biochemical analysis of C.
coronatus isolates revealed presence of toxic proteins in conidia and hyphae of the species. 30
kDa mycotoxin has been isolated from the culture filtrate /BOGUŚ and SCHELLER 2002/.
The fungus can be easily isolated on variety of simple media /GUSTAFSSON 1969, BAŁAZY
1993, GINDIN and BEN-ZE’EV 1994/. Utilization of the species for biological control is limited
by the fact that it might cause a human phycomycosis, rhinoentomophthoromycosis.
27. Conidiobolus obscurus (Hall et Dunn) Remaudière et Keller, Mycotaxon 11: 330,
1980
Bas.: Entomophthora obscura Hall et Dunn, Hilgardia 27: 162, 1957
Entomophaga obscura (Hall et Dunn) Batko, Bull. Acad. Pol. Sci., Ser. Sci. Biol. 12: 404, 1964c
Conidiobolus subgenus Conidiobolus obscurus (Hall et Dunn) Remaudière et Keller in Bałazy,
Flora of Poland. Fungi (Mycota) 24: 73-74, 1993
Syn.: Empusa thaxteriana Petch, Trans. Brit. Mycol. Soc. 21: 34, 1937 (nom. inval.)
Entomophthora thaxteriana (Petch), Hall et Bell, J. Insect. Pathol. 5: 186, 1963
Entomophaga thaxteriana (Petch) Batko, Bull. Acad. Pol. Sci., Ser. Sci. Biol. 12: 404, 1964c
Empusa planchoniana Thaxter, Mem. Boston. Soc. Nat. Hist. 4: 165, 1888, (nec Entomophthora
planchoniana Cornu, Bull. Soc. Bot. France 20: 189-190, 1873)
Entomophthora ignobilis Hall et Dunn, Hilgardia 27: 162, 1957
This pathogen was first discovered on numerous aphids of several genera by Thaxter in
Maine (the USA) in 1888. He, however, incorrectly identified the fungus as Empusa
planchoniana (Cornu) Thaxter /MACLEOD and MÜLLER-KÖGLER 1973/. PETCH /1937/, who
discovered the mistake, renamed the fungus observed by Thaxter as Empusa thaxteriana
Petch. After examination the Thaxter’s and Petch’s materials, HALL and BELL /1963/
concluded that E. thaxteriana and Entomophthora ignobilis, a pathogen described on the
aphid T. maculata by HALL and DUNN /1957/, were the same and the correct name for the
species was Entomophthora thaxteriana (Petch) Hall and Bell. BATKO /1964c/ subsequently
transferred the species to the genus Entomophaga. Several years later HUMBER /1978/ during
his studies on the identities of some Entomophthora species revealed that the name of
Entomophthora thaxteriana was not validly published and was rejected by him as
nomenclatural error because Petch provided neither the Latin description nor reference to a
previously published Latin diagnosis. Humber suggested Entomophthora ignobilis Hall et
Dunn to be the correct name for the fungal pathogen. In later study, REMAUDIÈRE et al. /1979/
demonstrated the synonymy of E. thaxteriana (E. ignobilis) and Entomophthora obscura, the
species described on T. maculata simultaneously with E. ignobilis by HALL and DUNN /1957/.
KELLER and REMAUDIÈRE /1980/ consequently transferred E. obscura to the genus
Conidiobolus what was later confirmed with nuclear cytology of the species /HUMBER 1981/.
C. obscurus is a very common and widespread pathogen specific only for aphids. It has
been found in a number of countries including e.g. the USA, Sweden, Finland, Norway,
Iceland, Great Britain, France, Switzerland, Poland, Slovakia, Russia, Australia, Egypt, or
South Africa /e.g. GUSTAFSSON 1969, THOIZON 1970, WILDING and PERRY 1980, MILNER and
SOPER 1981, PAPIEROK and HAVUKKALA 1986, KELLER 1987a, FENG et al. 1990, BAŁAZY
1993, BEHRENS 1993, VORONINA 1997, HATTING et al. 1999a, 2000, ŠTALMACHOVÁ and
CAGÁŇ 2000, NIELSEN et al. 2001b, ABDEL-MALLEK et al. 2003, KLINGEN and JAASTAD
2003/. C. obscurus demonstrates tendency to cause epizootics, but not as markedly when
compared with the most common aphid pathogen Pandora neoaphidis /KELLER and SUTER
1980/. Besides other aphid species, the pathogen is effective within populations of cereal
30
aphids /e.g. FENG et al. 1990, STEENBERG and EILENBERG 1993, HATTING et al. 1999a, 2000,
NIELSEN et al. 2001a/, pea aphid /e.g. WILDING 1975, BEHRENS 1993, CAGÁŇ and BARTA
2001/, cabbage aphid /SIVČEV 1991, 1992/, or black bean aphid /GUSTAFSSON 1969,
STEENBERG and EILENBERG 1993/.
The fungus has been widely studied as a potential aphid control agent /SOPER et al. 1975,
LATGÉ 1980, LATGÉ et al 1983, ČUDARE 1990, VORONINA 1997/, although a part of older data
also concerns the species Conidiobolus thromboides as a result of misidentification /HUMBER
et al. 1977/.
The culture of this pathogen can be established on Sabouraud’s dextrose agar or media
based on egg yolk and milk /KELLER 1987a/.
28. Conidiobolus thromboides Drechsler, J. Wash. Acad. Sci. 43: 38, 1953.
Syn.: Entomophthora virulenta Hall et Dunn, Hilgardia 27, 1957
Culicicola virulenta (Hall et Dunn) Batko, Bull. Acad. Pol. Sci., Ser. Sci. Biol. 12: 404, 1964c
Bas.: Conidiobolus subgenus Conidiobolus thromboides Drechsler in Bałazy, Flora of Poland. Fungi
(Mycota) 24: 72, 1993
C. thromboides is best known as an aphid pathogen, but it was first described as a

saprophyte from plant detritus in the USA /DRECHSLER 1953/. Few years later, another
pathogen, Entomophthora virulenta, was described from the aphid Therioaphis maculata
(Buckton) in California (the USA) /HALL and DUNN 1957/. BATKO /1964c/ transferred E.
virulenta to its rather controversial genus Culicicola. However, the Batko’s inclusion of E.
virulenta to Culicicola was based on the erroneous description of this species as producing
rhizoids /HUMBER et al. 1977/. Subsequently, morphological, biochemical and pathogenic
characters were used to compare C. thromboides and E. virulenta. Results of these analyses
gave evidence about a synonymy of both species /LATGÉ et al. 1980/.
The species has been recorded in Europe, the North America, Asia, Australia, the North
and South Africa and isolated from diverse sources including soil detritus and often dead
insects, mostly aphids /e.g. GUSTAFSSON 1969, THOIZON 1970, MILNER and SOPER 1981,
CHENG and LONG 1987, FENG et al. 1990, BAŁAZY 1993, STEENBERG and EILENBERG 1995,
HATTING et al. 1999a, 2000, NIELSEN et al. 2001a, ABDEL-MALLEK et al. 2003/. A number of
laboratory or field-plot tests have been conducted to assess of C. thromboides as a biological
control agent for pestiferous aphid species /e.g. HALL and DUNN 1958, MILNER and SOPER
1981, WILDING 1981, RAJ and RAMAKRISHNAN 1982, ČUDARE 1990, HATTING et al. 1999b/.
Methods for a mass-production of resting spores of C. thromboides were also developed
/MATANMI and LIBBY 1976, LATGÉ et al. 1978a,b, ČUDARE 1990/.
Entomopathogenic strains of this fungus misidentified as Entomophthora thaxteriana were
in the seventies of the last century widely studied in order to apply them in biological control
of aphids /HUMBER et al. 1977/.
The species showed good growth on Sabouraud’s agar /GUSTAFSSON 1969/.
29. Conidiobolus osmodes Drechsler, Am. J. Bot. 41: 571, 1954

Bas.: Conidiobolus subgenus Conidiobolus osmodes Drechsler in Bałazy, Flora of Poland. Fungi
(Mycota) 24: 71, 1993
This species was originally described as a saprophyte on plant detritus by DRECHSLER

/1954/. Later it was found to be an occasional pathogen of aphids in France /REMAUDIÈRE et
al. 1976b/. BEN-ZE’EV and KENNETH /1980/ found resting spores in larvae of Hypera postica
Gyll. in Israel and succeeded to isolate C. osmodes from the infected larvae, which proved to
be pathogenic to artificially infected Hypera larvae and houseflies. While the fungus was
31
recorded as an occasional pathogen of aphids in France, it was recorded as a very common

and destructive pathogen of Hypera spp. in Israel, even considerably more than other
pathogens in the fields /BEN-ZE’EV and KENNETH 1980/. In France, C. osmodes was found on
a few aphid species during cool weather and was able to occasionally manifest itself in the
populations at epizootic levels. An epizootic caused by the fungus was observed in a
population of Macrosiphoniella oblonga (Mordvilko) infesting chrysanthemums /PAPIEROK
and COREMANS-PELSENEER 1980/. C. osmodes is considered to be the only saprophytic
species of the genus Conidiobolus that could play an important role in the regulation of insect
populations, whereas the isolation of other saprophytic examples of Entomophthorales from
dead insects is accidental /PAPIEROK 1986/. In literature there are several records of C.
osmodes from aphid hosts. It was isolated from R. padi and S. avenae /LATGÉ 1975,
REMAUDIÈRE et al. 1981/ and observed on S. avenae and M. persicae in France /REMAUDIÈRE
et al. 1976b/, the cabbage aphid in Yugoslavia /SIVČEV 1991/, and undetermined aphids in
Poland /BAŁAZY 1993/. The species has been recorded in the North America, Australia, Israel
and Europe, isolated from organic detritus and dead insects, mostly Hypera spp. larvae and
aphids /BAŁAZY 1993/.
The culture can be established on “Entomophthora complete medium (ECM)” /BEN-ZE’EV
and KENNETH 1980/.
30. Conidiobolus destruens (Weiser et Batko) Ben-Ze’ev, Mycotaxon 14: 289, 1982
Bas.: Entomophthora destruens Weiser et Batko, Fol. Parasitol. 13: 144-149, 1966
Conidiobolus subgenus Conidiobolus destruens (Weiser et Batko) Ben-Ze’ev in Bałazy, Flora of
Poland. Fungi (Mycota) 24: 65-66, 1993
The fungus was described as Entomophthora destruens Weiser et Batko and found on
hibernating mosquitoes in natural shelters, caves, and wine cellars of Southern Moravia
/WEISER and BATKO 1966/. After a detailed study of karyological characteristics of species
recorded earlier under the generic name Entomophthora s.l., E. destruens was transferred to
Conidiobolus /BEN-ZE’EV 1982/.
C. destruens seemed to be rather host-specific to Culex pipiens Linné because in the
caves it was infected, whereas Culiseta annulata (Schrank) and Anopheles maculipennis
Meigen were not /MACLEOD and MÜLLER-KÖGLER 1973/. In some localities the fungus
destroyed more than 85% of the mosquitoes. Identical cases were later demonstrated in other
localities in Moravia, Bohemia, Slovakia, the Netherlands, Denmark, England and France
/MACLEOD and MÜLLER-KÖGLER 1973, MIETKIEWSKI and VAN DER GEEST 1985, EILENBERG
2002/. That opinion about strong specificity of C. destruens to C. pipiens was, however, later
displaced by the fact that strains of the fungus successfully infected in laboratory conditions
two aphid species, Megoura viciae Buckton and Aphis fabae /KREJZOVÁ 1972a/, two
members of Isoptera /KREJZOVÁ 1972b/, and LEPIDOPTERA /Krejzová 1971a/. That was the
only record of C. destruens from aphids. Application of conidia to M. viciae and A. fabae
resulted in 35% or 54% mortality, respectively /KREJZOVÁ 1972a/.
The pathogen is distributed in Central and Western Europe on C. pipiens and can be
isolated on egg yolk /WEISER and BATKO 1966, BAŁAZY 1993/.
Form-genus: Tarichium Cohn, Beitr. Biol. Pflanz. 1: 58-86, 1870

Syn.: Entomophthora (Tarichium) MacLeod et Müller-Kögler, Mycologia 62: 33-66, 1970
This genus was erected and typified with Tarichium megaspermum Cohn to include
species known only by their resting spores /COHN 1870/. The genus Tarichium, characterized
by smooth-walled or warted azygospores and inability to produce conidia, was later accepted
32
3. REVIEW OF LITERATURE: 3.5 Biology and ecology of Entomophthorales
by NOWAKOWSKI /1883/. THAXTER /1888/ did not regarded Tarichium as a genus but merely
as a group of Empusa species with unknown conidial stage and LAKON /1919/ proposed to use
the name Tarichium as a “temporary-auxiliary genus” for species known only from their
resting spores. MACLEOD et MÜLLER-KÖGLER /1970/ placed Tarichium as a subgenus to the
genus Entomophthora. However, this was not accepted /BEN-ZE’EV and KENNETH 1982a/. At
present Tarichium as a form-genus is generally admitted and considered to be a provisional
group without nomenclatural validity, since it has been applied for the description of species,
parasites of insects and mites, known only from their resting spores. Conidial stage of these
species has been still unknown, but is presumed to exist /BEN-ZE’EV and KENNETH 1982a,
HUMBER 1989, KELLER 1991, 1999, BAŁAZY 1993/. The genus includes 37 entomophagous
species with one parasitic to aphids /BAŁAZY 1993/.
31. Tarichium atrospermum (Petch) Bałazy, Flora of Poland, Fungi (Mycota) 24: 256-
257, 1993
Bas.: Entomophthora (Tarichium) atrosperma Petch, Trans. Br. Mycol. Soc. 17: 172, 1932
Entomophthora (Tarichium) atrosperma (Petch) MacLeod et Müller-Kögler, Mycologia 62: 39,
1970
Species was observed on unidentified aphid species in England and is known only from the
type locality /BAŁAZY 1993/.
3.5 BIOLOGY AND ECOLOGY OF ENTOMOPHTHORALES

Although, it is at present generally accepted that the fungi can invade and kill arthropod
hosts, the mechanism involved in this process was formerly open to speculation. For example,
in 1892 Cooke believed that fungal spores were ingested during feeding of the host and that
germination and infection only occurred within the host /SAMSON et al. 1988/. Now, it is
known that most entomopathogenic fungi invade their hosts via the cuticle and that infection
from ingested spores is of rare occurrence /e.g. BATKO 1974, BAŁAZY 1993, HAJEK and ST.
LAGER 1994/. Since the second half of the 20th century much work has been done to
understand all aspects of biology and ecology of Entomophthorales /for review see PELL et al.
2001/. For successful exploitation of these fungi in biocontrol a comprehensive understanding
of its biology and ecology is essential.
3.5.1 LIFE CYCLE OF ENTOMOPHTHORALES

A life cycle of Entomophthorales is complex and usually at least two types of spores are
involved (conidia and resting spores) within the cycle. A schema of basic life cycle is shown
in Figure 3. This schema includes both asexual conidia and sexual azygospores/zygospores.
However, it is necessary to reflect that there are many exceptions to the basic pattern.
Conidia, asexual spores, are fungal structures, which are responsible for infection initiation in
hosts and infection spreading during the season when hosts are active. Conidia are formed
apically on conidiophores, which have emerged through epidermis and cuticle of hosts and
have formed a hymenial layer covering dorsal and lateral parts of aphid’s abdomen /BATKO
1974, BROBYN and WILDING 1977, BUTT et al. 1990/. The fully developed conidia are
actively discharged out of conidiophores by hydrostatic pressure and spread by air current in
the environment /BATKO 1974/. However, some exceptions can occur in non-aphidophagous
Entomophthorales and conidia can be passively discharged in some genera, e.g. in genus
Massospora /SOPER et al. 1976/ or Strongwellsea /HUMBER 1997/. If conidia land on non-host
surface, they can produce replicate conidia (conidia of higher orders), i.e. a primary
33
Figure 3. General schema of life cycle of Entomophthorales
___________________________________________________________________________
a- resting spores forming germ conidia, b- primary conidia producing replicate ones, c- hyphal bodies in host’s
body, d- rhizoids attaching the host to a substrate, e- conidiophores with producing primary conidia, f- mature
primary conidia, g- primary conidia producing replicate ones, h- formation of resting spores in aphid’s body.
conidium originating from a dead host may produce a secondary conidium and a secondary
conidium may produce a tertiary conidium. Theoretically the process could continue until
vitality of protoplasm is exhausted or a susceptible host is encountered. If conidia land on host
surface, they germinate and germ tubes grow through host’s integument /BATKO 1974/. With
some genera, e.g. Neozygites, Zoophthora /GLARE et al. 1985, LATGÉ and PAPIEROK 1988/ or
Entomophthora /EILENBERG et al. 1995/, primary conidia are less or not infective and
secondary conidia, the infective structures, are always produced. The infective conidium
infects a new host by direct penetration of integument. Once the fungus has penetrated the
cuticle and epidermis, it starts to multiply as protoplasts, cell-walled hyphal bodies or
coenocytic hyphae in hemolymph and attacks host tissues /BATKO 1974, BROBYN and
WILDING 1977, BUTT et al. 1990/. The life cycle of Entomophthorales can follow one of two
paths. An asexual path where a host dies and conidiophores producing conidia emerge on the
host’s surface, or a path where resting spores are formed inside the host body. The fungi
complete the infection cycle within a few days, depending on temperature, host, and pathogen
species. After a host’s death conidiophores emerge on host’s surface and conidiogenesis starts
resulting in a production of infective units, conidia /BATKO 1974/. Resting spores are the
most important way that Entomophthorales survive periods when hosts are not present or
active. Resting spores are formed inside the aphid’s body /BAŁAZY 1993/, although certain
exceptions may occur, e.g. in Z. anhuiensis /LI 1986/. Resting spores may be sexual or
asexual, depending on fungal species. Some species were not observed to produce resting
spores at all. Moreover, there are species that are only known in their resting spore stage
/BALAZY 1993/. The soil environment is a main reservoir of resting spores in nature
/COREMANS-PELSENEER 1981, NIELSEN et al. 2003/. After a period of inactivity the spores
germinate and produce infective conidia, which may initiate infection in host populations. In
most cases, the resting spores are a dormant stage and require environmental stimulation for
germination /BITTON et al. 1979/.
In the ontogenesis of entomopathogenic species from the order Entomophthorales six main
phases can be distinguished /MADELIN 1968, BATKO 1965 in BATKO 1974/:
Infection phase – Infection takes place through integument of host. A fungal conidium sticks
to the insect cuticle, germinates, and perforates the cuticle by mechanical and enzymatic
action.
34
Lipolytic phase and localized development – After penetration into the host organism the
fungus at first grows slowly and does not spread within the insect body. At this time the
parasite secretes very active lipases, which very actively digest fats. This part of ontogenesis
is regulated genetically in some species and by environmental factors in others.
Colonization of host body – The fungus rapidly spread within the host body because of the
rapid proliferation of numerous hyphal bodies. As a result, the fungus penetrates into all
tagmata of the host’s body irrespective of the infection site. During this phase the fungus
utilizes nutrients that were accumulated in part during the previous phase and from the host’s
hemolymph without actively digesting the host’s tissues.
Proteolytic phase – After colonization of the host’s body, the fungus secretes very active
proteolytic enzymes. It penetrates into muscles and other tissues of insect and very rapidly
destroys the whole contents of host’s body. Only the chorion of mature eggs in bodies of
females resists the destructive action of proteolytic enzymes. These eggs are often fully
viable, a fact which indicates the impermeability of their integuments to fungal proteases.
Phase of host’s body mummification – After completion of proteolysis, hyphal bodies of
fungus suspended in fluid contents of the host’s body rapidly absorb the surrounding liquid
and grow quickly to form a spongy material. The consistency of the body changes to rigid.
Sporulation – Fungal hyphae breach the host’s integument, grow outside and are transformed
into conidiophores with conidia, or hyphal bodies conjugate in pairs and the fungus forms
thin-walled resting spores.
3.5.1.1 Initiation of pathogenesis

Conidia are the only infection units of Entomophthorales. Vegetative hyphae, even if in
direct contact with a susceptible host, are not infective /HUMBER 1990/. Conidium can be
defined as an asexual, unicellular, thin-walled infective unit containing one or more nuclei
/MACLEOD 1963, BEN-ZE’EV and KENNETH 1982a/. Infection begins by a germ tube
outgrowing from an infective conidium and penetrating the host’s integument /e.g. BROBYN
and WILDING 1977, KING and HUMBER 1981, BUTT et al. 1990, HAJEK and St. LAGER 1994/.
In appropriate conditions (e.g. high humidity, available nutrients on a cuticle surface, factors
necessary for a recognition of susceptible host) a discharged conidium after landing on a
suitable substrate germinates to form a germ tube. In unsuitable conditions the infection can
be aborted and the conidium may form a replicate conidium. A primary conidium discharge a
secondary conidium, which may in turn discharge a tertiary conidium, and so on /e.g.
BROBYN and WILDING 1977, KING and HUMBER 1981, EILENBERG 2002/. P. neoaphidis
conidia rapidly loss infectivity at 100% relative humidity on cover glass due to exhaustion of
energy reserves from production of the successive generations of conidia in a short time
/BROBYN et al. 1987/. Some genera produce secondary conidia more or less similar to the
primary ones, e.g. Pandora, Erynia, Entomophthora /e.g. BEN-ZE’EV and KENNETH 1982a,
HUMBER 1997, KELLER 1999, EILENBERG et al. 1995/, other genera, e.g. Neozygites or
Zoophthora, produce capillary conidia, mostly almond-shaped spores borne always on long
tapering capillary conidiophores /e.g. BAŁAZY 1993, KELLER 1997/. The capilliconidia are not
actively discharged /BAŁAZY 1993, KELLER 1997/. Some Conidiobolus species produce
microconidia, small globose conidia similar to primary ones in shape and produced on
radially projecting microconidiophores outgrowing from primary conidia. Microconidia are
best known in C. coronatus, in which 5 to 28 microconidia may be formed per a primary
conidium /PRASERTPHON 1963/. Not all C. coronatus strains can produce microconidia /KING
1976a/. Sometimes, primary conidia are regarded as dispersal fungal structures only infecting
when conditions are favourable and secondary (replicate) ones are the main infective
structures /GLARE et al. 1985, LATGÉ and PAPIEROK 1988, EILENBERG 1995, 2002/. Conidia
of the entomophthoralean pathogens are covered by adhesive gelatinous mucilage /BREY et al.
35
1986, BOUCIAS and PENDLAND 1991/, which is mainly responsible for the attachment of spore
to the integument /LATGÉ and PAPIEROK 1988, BOUCIAS and PENDLAND 1991/. While in
Conidiobolus genus, the mucilaginous material is present on the conidial surface /LATGÉ et al.
1986a/, in the genus Entomophthora the material is present between the outermost and
innermost conidial wall layers /EILENBERG et al. 1986/. Infective secondary capilliconidia in
genera Zoophthora and Neozygites also called non-adhesive spores are not covered with
adhesive material, but at their apices a drop of sticky liquid called haptor is produced /KELLER
1997/. In this case, the conidia adhere to the host’s cuticle by means of the sticky haptors
/GLARE et al. 1985/. Generally, all areas of aphid cuticle are susceptible to a conidial adhesion
/BREY et al. 1986/. The conidia stuck to the host’s surface may germinate forming penetration
hyphae. Conidia germinate within 2-4 hrs post-inoculation (at 20°C and saturated humidity)
/BROBYN and WILDING 1977/ and only one viable germ tube may develop per conidium,
however, some conidia give rise to several abortive germ tubes or germ initials /KREJZOVÁ
1977, BREY et al. 1986/. In most genera, e.g. Zoophthora, Conidiobolus, Pandora, Erynia,
Neozygites, or Entomophaga, a penetration of host cuticle is preceded by formation of
appressorium, from which an infection peg penetrates into the integument /BROBYN and
WILDING 1977, HYWEL-JONES and WEBSTER 1986, BUTT 1987, BUTT et al. 1990,
MAGALHÃES et al. 1990a,b, 1991, WRAIGHT et al. 1990, NADEAU et al. 1996a, DARA and
SEMTNER 1998, HAJEK et al. 2002b/. However, a penetration with some species, e.g. P.
neoaphidis and C. obscurus, can also occur without the production of appressoria /BROBYN
and WILDING 1977, BREY et al. 1986/. The appressorial formation has not been reported in
studies of species from the genera Entomophthora and Batkoa /BROBYN and WILDING 1977,
LAMBIASE and YENDOL 1977/. Appressoria are usually produced in response to the
recognition that the substrate is suitable for infection /BUTT et al. 1990/ and they represent an
adaptation for concentrating physical energy and lytic enzymes over a very small area to
make subsequent penetration more efficient /ST. LEGER 1991, HAJEK and ST. LEGER 1994,
BECHINGER et al. 1999/. Formation of appressoria from P. neoaphidis conidia was
significantly higher on living substrates than on inert cover glass /DARA and SEMTNER 1998/.
On the other hand it was observed that higher percentages of appressoria were formed from
conidia of Entomophaga maimaiga Humber, Shimazu et Soper deposited on hard surfaces
such a polystyrene than on water agar, and no nutrients or chemical stimuli were required for
appressorial formation, however, nutrients could stimulate growth of germ tubes /HAJEK et al.
2002b/. Not only a growth of germ tubes, but also germination of conidia is regulated by
various nutritional and physicochemical factors /KERWIN 1982, UZIEL and KENNETH 1999/.
For instance, free aminoacids and monosacharides originating from the honeydew and
aqueous cuticular extracts of A. pisum favour the formation of germ tubes in conidia of C.
obscurus /SAMPEDRO et al. 1984, BOUCIAS and LATGÉ 1988/. Host cuticular lipids stimulate
an appressorium and penetration tube formation of Erynia conica on a susceptible host and do
not inhibit a conidial resporulation on a non-susceptible host /NADEAU et al. 1996a/.
Moreover, certain self-inhibited germination resulting from a high spore concentration on
host’s cuticle was observed for C. obscurus /BREY et al. 1986/. It was hypothesized that the
self-inhibition of conidial germination due to crowding could be caused by the CO 2 produced
when conidia respire /COOK and WHIPPS 1993/. However, this phenomenon of self-inhibited
germination was not observed for Entomophaga maimaiga conidia deposited on larval cuticle
/HAJEK et al. 2002a/ or Erynia conica conidia on microscope slides /NADEAU et al. 1996b/,
conversely a percent germination was positively associated with conidial density on water
agar /HAJEK et al. 2002a/. Germ tube penetrates the aphid integument mechanically and
enzymatically. Lipases, proteases, and chitinases were reported being produced exocellularly
by the fungi /e.g. GABRIEL 1968, BROBYN and WILDING 1977, KING and HUMBER 1981,
LATGÉ et al. 1982, BREY et al. 1986, LATGÉ and PAPIEROK 1988/. A strategy for the cuticle
36
penetration seems to be various depending on particular species. At a penetration site a

circular entry may be formed, e.g. P. neoaphidis and Z. radicans /BUTT et al. 1990, WRAIGHT
et al. 1990/, or tetraradiate fissure, e.g. C. obscurus /BROBYN and WILDING 1977/, and finally
triradiate penetration may be formed, e.g. genus Entomophthora /BROBYN and WILDING 1977,
EILENBERG et al. 1995/. Generally, a penetration is most frequently observed on the abdomen,
to a lesser extent on the thorax, and never on the head or appendages /BREY et al. 1986/. It is
of interest that number of penetrating germ tubes is often low. Germ tubes may grow errantly
over the surface of cuticle and do not penetrate it /BROBYN and WILDING 1977, BREY et al.
1986/. In C. obscurus less then 10% of germinating conidia penetrated a host’s cuticle /BREY
et al. 1986/.
3.5.1.2 Invasion and colonisation processes during pathogenesis

As soon as the fungus has breached the cuticle and has reached the haemocoele, it begins
to multiply and invade the host’s tissues /BROBYN and WILDING 1977, KOBAYASHI et al.
1984, BUTT et al. 1990/. In case of, for instance, C. obscurus the invading hyphae separate to
form large hyphal bodies, which subsequently start to multiply by budding /LATGÉ 1980/.
Hyphal bodies are single, coenocytic cells, which vary in shape from spherical (e.g. N.
fresenii) to hyphae-like (e.g. P. neoaphidis), proliferate rapidly, and circulate readily in
hemolymph to all parts of infected host /BROBYN and WILDING 1977, KOBAYASHI et al. 1984,
HUMBER 1990/. However, most entomophthoraceous and neozygitaceous species develops as
wall-less protoplasts within the first stages of pathogenesis and later differentiate to walled
hyphal bodies /e.g. MACLEOD et al. 1980, KOBAYASHI et al. 1984, BUTT et al. 1981, 1990,
KELLER 1997/. These pathogens probably switch to the protoplast form as a response to
contact with the nutrient rich hemolymph /BUTT et al. 1990/. The protoplasts may vary in size
and shape and they multiply by budding /BUTT et al. 1981/. Wall-less protoplasts are assumed
to be important in the early stages of infection, because these structures are undetectable by
host’s defence responses /DUNPHY and NOLAN 1982, BREY et al. 1986/. The protoplastic
behaviour by entomopathogens also offers other possible advantages. (1) The protoplastic
form facilitates rapid fungal growth. Contrary to walled hyphal bodies, the protoplasts feed by
pinocytosis and so they feed more efficiently. Moreover, (2) energy for rapid multiplying is
not expended in cell wall synthesis /BUTT et al. 1981/. The first hyphal bodies/protoplasts are
usually observed in a fat body /BROBYN and WILDING 1977, BUTT et al. 1981, KOBAYASHI et
al. 1984, FUNK et al. 1993/. The attachment of fungal structures to the fat body is probably
due to the high concentration of energy found there. The fat body is a source of nutrients for
initial fungal growth /FUNK et al. 1993/. Shortly after the fungus has established in the host,
most tissues are digested, but usually tracheoles, gut, and embryos are only marginally
affected /BROBYN and WILDING 1977, BUTT et al. 1990/. It is assumed that the protoplastic
form lacks or ceases a production of enzymes required to digest the cuticle, and so the cuticle-
enveloped tracheoles, gut, and embryos are only slowly digested /BUTT et al. 1990/. Most
entomopathogens depend on the continual feeding of the host to provide the water and
nutrients required by both host and pathogen /HUMBER 1990/. When the host’s body is
occluded and nutrients in hemolymph are exhausted, the protoplasts regenerate a wall /BUTT
et al. 1990/. In moribund aphids, the pathogens fill the body cavity and attack any organs and
tissues that remained unaffected through the earlier developmental stages /BROBYN and
WILDING 1977, BUTT et al. 1990, HUMBER 1990/. Hyphal bodies differentiate into rhizoids,
pseudocystidia, and conidiophores, however, the factors that control the differentiation have
been still unknown /BROBYN and WILDING 1977, BUTT et al. 1990/. This differentiation is
preceded by a hyphal body concentration beneath the cuticle and orientation perpendicularly
to a surface /BUTT et al. 1990/. Rhizoids emerge shortly before or after the host’s death and
prior to conidiogenesis /BROBYN and WILDING 1977, KING and HUMBER 1981/. They are
37
formed from large, vacuolated hyphal bodies in the mid-ventral region of the insect /BUTT et
al. 1990/. Rhizoids are broad, highly vacuolated, sterile hyphae ending in a more or less
complex holdfast or series of short adhesive appendages anchoring an infected aphid to a
substrate /KING and HUMBER 1981/. Rhizoids secrete a viscous substance /MACLEOD 1963/
and adhere firmly to a plant cuticle, but do not penetrate it /BROBYN and WILDING 1977,
BUTT et al. 1990/. Not all species form rhizoids, for instance species of genera Conidiobolus
and Neozygites are known being non-rhizoidal /KELLER 1987a, 1991, 1997/. However, C.
obscurus was reported to form a few delicate rhizoids around mouthparts and forelegs
/BROBYN and WILDING 1977/.
A lethal time of infected hosts is generally within a few days, depending on fungal species,
strain, insect host, and incubation temperature /MILNER and BOURNE 1983, SCHMITZ et al.
1993/. Adult aphids infected with Entomophthorales die 3-6 days after inoculation (at 20°C)
/WILDING 1969, 1971, DEAN and WILDING 1971/. 24 hrs (at 25°C) after inoculation with P.
neoaphidis, aphids are moving normally. But 36 hrs post-inoculation, aphids become slow-
moving and then soon become immobile. At this infection stage hyphal bodies are still wall-
less, but the cell wall formation has started. Aphids usually die between 60 and 96 hrs after
inoculation. 24 hrs after death, cadavers change in colour and cell walls are completely
regenerated. 24 hrs after the colour change, the hyphae penetrate through the cuticle to form
conidiophores /BROBYN and WILDING 1977, KOBAYASHI et al. 1984, BUTT et al. 1990/.
Host’s death is probably caused by destruction of tissues due to secretion of digestive
enzymes, physiological starvation of the host when the fungus has consumed all reserves, or
one or more mycotoxins may be produced that kill the host before the haemocoele may have
been filled with fungal hyphae /LATGÉ et al. 1980, PAPIEROK and COREMANS-PELSENEER
1980, ROBERTS and HUMBER 1981, HALL and PAPIEROK 1982, GILLESPIE and CLAYDON
1989, ČUDARE 1990/.
3.5.1.3 Post-mortem processes of pathogenesis

Post-mortem stage of infection includes a development of sterile structures like
pseudocystidia and rhizoids as well as reproductive units like conidia and/or resting spores.
Shortly after the death of insect, unbranched or variously branched conidiophores grow out
from hyphal bodies and emerge through exoskeleton covering most part of insect body except
the mid-ventral region. Conidiophores breach the relatively thin cuticle of abdomen and
intersegmental membranes, but not the thick cuticles of head and thorax. They break out
through the host cuticle using a combination of enzymatic and mechanical means. At a tip of
conidiophore a conidium starts to generate. Normally, as the protoplasm of conidiophore
passes into the developing conidium, the conidium enlarges until its mature shape and size is
attained. It is then separated from the conidiophore by a transverse septum /MACLEOD 1963,
BROBYN and WILDING 1977, EILENBERG et al. 1986, LATGÉ and PAPIEROK 1988, BUTT et al.
1990/. An important property of most entomophthoralean genera is ability to actively
discharge conidia. A discharge mechanism has been described with different terms /BEN-
ZE’EV and KENNETH 1982a, BAŁAZY 1993/. Passively detached are only secondary conidia:
capilliconidia of genera Neozygites, Zoophthora, Conidiobolus and Basidiobolus as well as
microspores of the genus Basidiobolus /BEN-ZE’EV and KENNETH 1982a/. BEN-ZE’EV and
KENNETH /1982a/ distinguished three different ways of conidial forcible discharge: a
“rounding off” characteristic for most genera, a “sporophore cannon” in the genus
Entomophthora, and a “sporophore rocket” known in the genus Basidiobolus. A discharge of
conidia in the genus Entomophthora was earlier described as a “sporophore canon” when
spores were discharged with a stream of cytoplasm from the conidiophore /INGOLD 1971,
MACLEOD et al. 1976, HUMBER 1981, BEN-ZE’EV and KENNETH 1982a/. However, in recent
studies, this theory has been rejected and “rounding off” mechanism of discharge has been
38
suggested /EILENBERG et al 1986, 1995/. A general pattern for conidial discharge is as follow:
Contents of fully developed conidium as well as conidiophore bearing the conidium absorb
water rapidly and osmotic pressure increases in both. However, at conidial maturity the
osmotic pressure is greater in the conidium thus a columella, by which conidium is jointed
with conidiophore, is forced into the conidiophore. The pressure exerted is so strong that the
outer of the two walls enclosing the conidium ruptures in a circle around its base and the
conidium is discharged into the air and carried a considerable distance /MACLEOD 1963/. The
conidia can be discharged several millimetres away of the cadavers /SIX and MULLENS 1996,
HEMMATI et al. 2001a/. The discharge intensity and discharge dynamics are dependent on
temperature. Within the range of 10-30°C, a higher temperature results in a more rapid
discharge of primary conidia /MILNER 1981b, GLARE et al. 1986a, MORGAN et al. 1995,
HEMMATI et al. 2001a/.
Pseudocystidia are mostly unbranched or slightly branched sterile hyphae enlaced among
conidiophores, but extend beyond the level of conidiophores /BROBYN and WILDING 1977,
KING and HUMBER 1981/. Their function has been a point of debate. They break through the
host’s cuticle before conidiophore arising and facilitate the emergence of conidiophores. It is
also assumed that pseudocystidia may help trap a layer of moist air over the body of infected
insect and thus prolong a period during which conidia are discharged /BROBYN and WILDING
1977/.
Most species are able to form resting spores inside host body. Two types of resting spores
are observed in Entomophthorales, zygospores or azygospores. Zygospores arise from
conjugation of two hyphal bodies (e.g. in N. fresenii), whereas conjugation is not evident in
the production of azygospores /KING and HUMBER 1981/. The spores are characterised by a
thick chitin-protein wall and contain a large oil droplet rich in saturated fatty acids /LATGÉ et
al. 1984a/. The wall has two thick layers and spore lie free within a thinner outer wall
(episporium). The episporium may be hyaline (e.g. C. obscurus) or coloured (e.g. genus
Neozygites), and smooth (e.g. Z. aphidis) or variously ornamented (e.g. C. coronatus) /KING
and HUMBER 1981/. The thicker inner layer of spore wall consists of three sub-layers of
different density and structure /ČUDARE 2001/. Matured resting spores of C. thromboides and
Z. radicans are binucleate, but in activated spores the number of nuclei is reduced to one by
fusion and before germination subsequent nuclear divisions result in a multinucleate germ
tubes /MCCABE et al. 1984/. Zygospores of the genus Neozygites are also binucleate /KELLER
1997/. The circumstances that give rise to the development of resting spores have been
imperfectly understood. It was observed that resting spores start to form toward the end of the
growing season /THAXTER 1888/, but they were also formed during summer /DUSTAN 1927/.
It is assumed that probably a lowering of temperature or a short period of dry weather at a
certain stage of infection development may supply a stimulus necessary for the formation of
these spores /MACLEOD 1963/. C. obscurus formed resting spores in aphid body at 2 and 6°C
and high humidity, but when the temperature was greater than 12°C, conidiophores and
conidia were formed on the surface of the cadaver /PAPIEROK 1978/. A physiological state of
host was also demonstrated that may stimulate initiation of resting spore formation. In non-
feeding cereal aphids, probably unsuitable for optimal fungal development, a production of
resting spores of Z. radicans was induced /FENG and JOHNSON 1991/. In nature, cadavers
filled with resting spores usually fall to the soil where they remain during the winter. Early
spring temperatures induce asynchronous germination of resting spores and infective conidia
are produced over a period of several weeks /LATGÉ and PAPIEROK 1988/.
3.5.1.4 Host’s defence responses to the pathogens

Insect immunity is a remarkable underexplored research area. Little is known of how
insects recognize a fungus and their immune responses are initiated. Commonly, there are two
39
fundamental defence mechanisms in insect organisms, the humoral and cellular responses
/RATCLIFFE and ROWLEY 1979, BUTT et al. 1988, HAJEK and ST. LEGER 1994/.
The humoral responses are usually characterised by a melanin deposition on an invading
organism. Localised cuticular melanization adjacent to fungal appressoria and penetration
pegs is often the first response detected in insects /BROBYN and WILDING 1977, BREY et al.
1986, BUTT et al. 1988, 1990, HUMBER 1990/. The melanization may alter the mechanical
properties of cuticle, making it both more rigid /ANDERSON 1985/ and more resistant to
enzymatic degradation /KUO and ALEXANDER 1967/. Melanin is known to inhibit microbial
enzymes /KUO and ALEXANDER 1967/ and is fungistatistic /SÖDERHÄLL and AJAXON 1982/.
However, the melanization often occurs too late or in insufficient magnitude to prevent fungal
infection /BUTT et al. 1988, ST. LEGER 1991/. Most penetration pegs are able to breach the
melanin collars, deposited around them, or grow faster than the melanin could be synthesized
and deposited. It was observed, that Z. radicans produced a further peg from appressorium,
after the initial infection peg had been completely arrested by a thick melanotic capsule /BUTT
et al. 1988/.
Granulomatous encapsulation by haemocytes is the major cellular immune response by
insects /RATCLIFFE and ROWLEY 1979, GÖTZ 1986/. During the encapsulation, granulocytes
are attracted to the fungus in the hemolymph and may engulf it in the process of phagocytosis.
Then plasmatocytes are recruited and form a pseudotissue thus differentiating a granuloma, in
which the fungus may be lysed /GÖTZ 1986/. In a matter of fact, the encapsulation may only
protect hosts against weakly virulent pathogens, whereas hypervirulent ones overcome the
encapsulation /HUNG et al. 1993/.
Defence responses against the entomophthoralean fungi are frequently restricted just to the
cuticular melanization at the sites of fungal penetrations and no obvious cellular immune
response mediated by haemocytes is observed /BROBYN and WILDING 1977, BREY et al. 1986,
BUTT et al. 1988, 1990/. It is because most Entomophthorales employ a unique method of
how to avoid cellular defence responses. They multiply during first stages of host colonisation
like wall-less protoplasts /MACLEOD et al. 1980, KOBAYASHI et al. 1984, BUTT et al. 1981,
1990, KELLER 1997/ and therefore do not attract haemocytes /DUNPHY and NOLAN 1982/.
Saccharides and other components secreted by a fungus for assembly into cell walls are the
key elicitors of insect immune responses /DUNPHY and NOLAN 1982, PENDLAND and BOUCIAS
1986, BEAUVIAS et al. 1989/.
A specific behaviour of insects may also act as an effective defence against potentially
lethal infections. For instance, grasshoppers and houseflies bask in the sun, which raises
internal body temperature above optimum of development of Entomophaga grylli (Fresenius)
Batko and Entomophthora muscae (Cohn) Fresenius, respectively, and the insects thereby
recover from infection (the “behavioural fever”) /CARRUTHERS et al. 1992, WATSON et al.
1993, KALSBEEK et al. 2001/. It was observed that a growth and sporulation of
Entomophthorales occurs in a range of temperature of about 10-25-(30)°C and upper
threshold lies in the range of about 25-38°C, depending upon species /e.g. HALL and BELL
1960, VORONINA 1968, ROBERTS and CAMPBELL 1977, MILNER and LUTTON 1983, PAPIEROK
1985, GLARE et al. 1986a, GALAINI-WRAIGHT et al. 1992/. However, this behaviour is not
definitely attributed to fungal action since this sun basking is considered the natural behaviour
of the insect /PELL et al. 2001/.
3.5.1.5 Winter survival of Entomophthorales

The thought that Entomophthorales may survive a period of adverse conditions for fungal
development in the form of thick-walled resting spores is generally accepted /e.g. BAŁAZY
1993, KELLER 1987a, 1991, 1997/. However, not all species have been documented to
produce the spores. Among the aphid pathogenic fungi resting spores have been described as
40
occurring in vivo for all species with except Z. phalloides, P. neoaphidis, P. kondoiensis, and
N. lageniformis /KELLER 1991, BAŁAZY 1993, NIELSEN et al. 2001a/. It is possible to assume
that the survival strategy depends on the fungus species and a single species may have more
than one possible means of survival available. Besides resting spores, other ways of survival
have been observed or supposed being existing, namely: specialized hyphal bodies /KELLER
1987b, FENG et al. 1992b/, conidia or “loriconidia” /WEISER and BATKO 1966, LATTEUR and
RANDAL 1986, NIELSEN et al. 2003/, or a survival through continuous conidial infections in
anholocyclic aphid populations /BYFORD and REEVS 1969, WILDING 1973, MCLEOD et al.
1998/.
The main reservoir of resting spores is soil /COREMANS-PELSENEER 1981/, but resting
spores may also be deposited on a tree bark /HAJEK et al. 1998/, or in dead aphids attached in
crown of trees /SOPER and MACLEOD 1981/. Thick-walled resting spores are not directly
infective, but they may germinate and form infective conidia /COREMANS-PELSENEER 1981,
HUMBER 1990/. Resting spores of most species, to germinate, have to undergo a period of
obligatory dormancy, which can be broken by a 2-4 month storage in humid cold conditions
/BITTON et al. 1979, PERRY and LATGÉ 1982, PERRY 1988, PERRY and FLEMING 1989,
NIELSEN et al. 2001a/. In some species short /BEN-ZE’EV et al. 1990/ or no dormancy was
observed /SOPER et al. 1975/. The breaking of dormancy is thought to be a very complex
process controlled by several factors /BEN-ZE’EV et al. 1990, NIELSEN et al. 2001a/. For the
population of spores as a whole, germination is asynchronous /HAJEK and HUMBER 1997/.
The timing of the spore germination seems to be correlated with host, pathogen, temperature,
humidity and light /WALLACE et al. 1976, PERRY and LATGÉ 1982, PERRY and FLEMING 1989,
BEN-ZE’EV et al. 1990, THOMSEN et al. 2001, NIELSEN et al. 2003/. Exact requirements for
initiation of germination have not been completely clarified. Not all resting spores germinate
during one year /HAJEK and HUMBER 1997/, thus creating a reservoir of resting spores in the
soil. For instance, resting spores of Entomophaga maimaiga are present in the soil about 11-
12 years after an epizootic /HAJEK et al. 2000/. Entomophthoralean resting spores have been
tested whether they respond to the presence of host organisms by their activation from
dormancy or stimulation to germination, but no such action has been found /HAJEK and
EASTBURN 2001/. On the contrary, results of NIELSEN et al. /2003/ supported the hypothesis
that germination of overwintering or quiescent inoculum is stimulated by host-induced
factors.
WEISER and BATKO /1966/ observed in C. destruens thick-walled conidia and described
them as “loriconidia”. They were formed from conidia by thickening their cell walls. And the
structures were supposed to substitute the function of resting spores. NIELSEN et al. /2001a,
2003/ observed conidia similar to those described as “loriconidia” produced on aphids, which
were killed with P. neoaphidis and stored for 1 month on sterilised soil in darkness at 5°C.
From in vitro cultures the thick walled conidia were observed as well.
LATTEUR and RANDALL /1986/ found out that primary conidia of P. neoaphidis were able
to preserve viability for 6-8 months at 5°C deposited on soil surface under dark conditions.
Conidia or hyphal bodies of P. neoaphidis deposited on soil surface were able to produce
replicate conidia or sporulate, depending on temperature, for 23-96 days and the inocula
retained the ability to initiate infections in aphids after storage for 14-64 days, depending on
temperature /NIELSEN et al. 2003/. However, conidia on plant leaves remain their infectivity
only up to 1 month at 5°C /SCHOFIELD et al. 1995/ or 2 weeks at field conditions /BROBYN et
al. 1985/.
Hyphal bodies of P. neoaphidis in aphid cadavers were able to survive a period of several
months under dry and cold conditions /WILDING 1973, COURTOIS and LATTEUR 1984,
LATTEUR et al. 1985/. FENG et al. /1992b/ found a peculiar spherical hyphal bodies
distinguishable from the regular ones. The spherical hyphal bodies appeared only in the
41
autumn and they were able to overwinter inside aphid cadavers and produce infective conidia.
P. neoaphidis survived the winter in the form of hyphal bodies on plant substrates above
ground in relatively dry environments, but not in the moist soil. Such a type of hyphal bodies
has also been documented for another aphid pathogen. In autumn, E. planchoniana produced
thick-walled hyphal bodies in colonies of Drepanosiphum acerinum (Walker) in Switzerland
even though resting spores are known from this species /KELLER 1987b/. In spring it was
possible to infect healthy aphids with E. planchoniana from aphids containing the thick-
walled hyphal bodies.
Finally, P. neoaphidis was also suggested that may survive winter month in anholocyclic
aphid populations via continuous conidial infections /BYFORD and REEVS 1969, WILDING
1973, REMAUDIÈRE et al. 1981, DARA and SEMTNER 2001/.
3.5.2 EPIZOOTIOLOGY OF ENTOMOPHTHORALES IN APHID POPULATIONS

3.5.2.1 Types of disease prevalence in aphid populations
A disease, expressed as an unbalanced state of physiological equilibrium of organism, can
be caused by numerous biotic and abiotic factors. The insect epizootiology concentrates on
infectious diseases brought about by infectious (biotic) agents; however there may be a
complex of factors, including environmental conditions, contributing or predisposing of insect
to a susceptibility to the biotic agents, thus they should be considered in epizootiology /FUXA
and TANADA 1987/.
The intensity and extent of the interaction between the host and pathogen populations
determine the state of disease prevalence that is, generally, epizootic or enzootic. The
definition of epizootic disease could be expressed by different ways. Simply can be said, it is
an unusually large number of cases of disease in host populations /BARR 1979/. However, to
know what is an unusually large number, it is necessary to have knowledge about a past
history of the disease for a number of years in a particular host populations /FUXA and
TANADA 1987/. Enzootic disease is a case of usually low prevalence of infections, which are
constantly present in populations. A certain sort of balance and coexistence between pathogen
and host is characteristic of enzootic disease /VAN DER PLANK 1975, FUXA and TANADA
1987/. While epizootics are sporadic, time-limited and characterized by a sudden change in
prevalence and incidence of case of infection, enzootic disease levels are of long duration and
a ratio of daughter to parent infections approximately equals one. During epizootics a ratio of
daughter and parent infections significantly exceeds one /VAN DER PLANK 1975/.
A clear-cut distinction between enzootic and epizootic state of disease prevalence in aphid
populations was presented by DEDRYVER /1983/. He classifies three types of mycoses in aphid
populations by putting into relationship a number of living aphids and aphids killed by
Entomophthorales in a population. In the first type, epizootic type, number of infected aphids
increases in populations more quickly than number of living aphids. The number of living
aphids diminishes and the number of infected aphids continues to increase to exceed a ratio of
infected/living aphids 1/1. In the second type of mycosis, enzootic type, number of infected
aphids remains approximately proportional to number of living aphids, both in the population
growth period and in their diminution phase. In most cases, the ratio of infected/living aphids
is 1/100 during the population growth phase and 1/10 during their reduction. In the third type
of mycosis, there is a growth then a reduction in the number of living aphids, while the
number of infected aphids remains constantly low.
STEINHAUS /1954/ divided the epizootic wave into three phases; the preepizootic phase, the
period just preceding the increase in numbers of infected insects; what results in the epizootic
phase itself, the climactic portion of epizootic wave; and followed by postepizootic phase,
which is characterised by the nature and number of the survivors.
42
3.5.2.2 Factors governing disease prevalence and determining disease character

The primary factors that are involved in the initiation and development of epizootics of
infectious diseases in insects are the pathogen population, host population, and efficient
means of pathogen transmission. Since the pathogen-host interaction exists within an
environment characterised by its abiotic and biotic properties, the elements of environment
also interfere in a disease development.
3.5.2.2.1 Host populations
There are a number of aphid population properties, which can determine a disease
character. The most significant in the epizootics of entomophthoralean diseases seems to be a
susceptibility of host population to pathogens, density or behaviour of insect population, and
relationships among different host populations in the environment /WATANABE 1987/.
Many reports point out differences between two or more isolated populations of insect
species in responding to fungal pathogens. With respect to aphids, PAPIEROK and WILDING
/1979/ reported respond differences in two clones of pea aphid to infection by fungus C.
obscurus. Likewise, MILNER /1982, 1985/ determined two biotypes of pea aphid with distinct
susceptibility to P. neoaphidis in Australia. A certain clonal variation of pea aphid in
resistance to P. neoaphidis was also found by FERRARI et al. /2001/. Evidently, a variety of
endogenous and exogenous factors are involved in aphid susceptibility, which may act
together, e.g. age, moulting, food and environmental factors. Several reports show an insect
larvae decrease in susceptibility to fungal infection as they age as well as not all stages of host
development are of even susceptibility to mycosis. Nymphs of pea aphid are more susceptible
to the fungal infection than adults and alate adults are more susceptible than apterous adults
/MILNER 1982, 1985, LIZEN et al. 1985/. Alate adults with age from 3 to 4 days are the most
resistant to infection /LIZEN et al. 1985/. Moulting may remove penetrating fungus prior to the
colonisation of insect if it occurs shortly after inoculation /VEY and FARGUES 1977, FARGUES
and RODRIGUEZ-RUEDA 1979/. Susceptibility demonstrated in the laboratory may not relate to
infection in the field, suggesting differences between physiological and ecological
susceptibility. As a matter of fact susceptibility relates both to the physiology of insect and to
its behaviour, which may encourage or discourage the acquisition of conidia /GOETTEL 1994,
ROY et al. 1998/. Concerning to aphid’s behaviour, it was observed that M. dirhodum and D.
noxia were predominantly infected by P. neoaphidis probably because the aphid species
inhabit more humid microenvironments in cereal crops such as undersurface of lower leaves
or tightly rolled recently developed leaves /DEAN and WILDING 1971, DEDRYVER 1983, FENG
et al. 1991, FENG et al. 1992a/. S. avenae primarily infesting the ears of cereals, the
microhabitat with lower humidity, was less infected by P. neoaphidis in comparison to C.
obscurus, which is more tolerant to lower humidity /WILDING 1969, DEAN and WILDING
1971, FENG et al. 1992a/. There is some evidence that even food quality is important in the
susceptibility of insect to parasitic fungi. RAMOSKA and TODD /1985/ found that different food
supplied to host (Blissus leucopterus Say) could influence indirectly a pathogen (Beauveria
bassiana (Balsamo) Vuillemin) development within hosts. Similarly, GOETTEL et al. /1993/
revealed that leafcutter bees, Megachile rotundata F., reared on natural provisions were
generally less susceptible to Ascosphaera aggregata Spiltoir et Olive as compared with bees
reared on artificial provisions. Bioassay with Z. radicans against Therioaphis trifolii Monell f.
maculata indicated that aphids that starved for 24 hr during inoculation with primary spores
were less susceptible than those that were removed from plants for just the period of exposure
to the primary spore shower /MILNER and SOPER 1981/. The results imply that during initial
stages of infection a highly nutritious environment is required for hyphal bodies to multiply
rapidly in the hemolymph. A study of fungi-parasitoids interference in aphid host revealed
43
that parasitoid developing inside the aphid’s body may predispose the host to the infection by
P. neoaphidis /POWELL et al. 1986b/.
A dissemination of pathogens within a host population or among populations plays an
important role in the development of epizootics. Besides mechanical factors, such as rain and
wind (see section 3.5.2.2.4), biotic factors, including population density, or behaviour of
infected individuals can also affect inoculum spreading. Aphids themselves may serve as
biotic agents for inoculum dispersal. Aphids may spread infection among colonies either in
the same crop or among different crops /WILDING and PERRY 1980, ČUDARE 1990/. Rather
aggregate populations typical of aphids facilitate a pathogen dispersal and thus promote the
rapid spread of disease /SOPER and MACLEOD 1981, HUMBER 1990/. High host densities in
colonies increase in a contact between uninfected and infected hosts and enhance the
probability of horizontal transmission of infection /STEINHAUS 1954, ČUDARE 1990/. Aphids
in dense patches on goldenrod stems were more vulnerable to N. fresenii infection than aphids
in sparse patches /CAPPUCCINO 1988/. There is also study about effect of fungal diseases on
spatial patterns of cereal aphids. It was found out no difference between spatial distribution of
infected and living aphids on cereals /FENG and NOWIERSKI 1992a/. Presence of alternative
hosts in insect community may contribute to increase in inoculum density and dispersal,
resulting in increased infection in primary hosts. Sympatric aphid colonies of A. pisum and A.
kondoi were observed being differentially infected by P. neoaphidis in California. While P.
neoaphidis killed few individuals of A. kondoi, the pathogen density increased in more
susceptible A. pisum. It was assumed that the low-level infections in A. kondoi might greatly
increase the inoculum available for transmission to the highly susceptible A. pisum
/PICKERING and GUTIERREZ 1991/. One could point out that not only non-target aphids in
insect communities may play a role of alternative hosts for Entomophthorales /STEENBERG
and EILENBERG 1995/. Low-specific fungi like Z. radicans, which has been isolated from
various insect orders, could be theoretically spread among different insect populations in the
environment. A cross-transmission of infection in laboratory is usually scarce and has not
been observed in nature /MILNER and MAHON 1985/. Adaptation of strains to a host
taxonomically related to the host from which the strains were isolated probably exists
/PAPIEROK et al. 1984/. However, isolates of the fungal species originated from non-
Homopteran hosts show their infectivity to aphids /PAPIEROK et al. 1984, MILNER and MAHON
1985/. KELLER and SUTER /1980/ showed that epizootics in aphid populations on several
crops began when a large proportion of aphids on weeds within and around the crop were
infected. The influence of weeds on aphid enemies including Entomophthorales was studied
in England, as well /POWELL et al. 1986a/. A competition between two aphid species via the
pathogen P. neoaphidis was also discussed /POPE et al. 2002/. Parasitic fungi act as density-
dependent mortality factors if the pathogens operate under favourable weather conditions
/STEINHAUS 1954, WATANABE 1987/ and correlation between fungal infection and host
density was found /WILDING and PERRY 1980, FENG et al. 1991/. Because the threshold host
density at which the fungi start to be effective is generally high, epizootics in aphid
populations usually occur at high host densities /SIVČEV 1991, PELL et al. 2001/. However,
this is not always true, for instance P. neoaphidis epidemic in pea aphid populations was
maintained at a density below 4 hosts per stem /PICKERING and GUTIERREZ 1991/. For
Schizolachnus piniradiatae (Davidson), a threshold level of 50-80 aphids per 30-cm branch of
pine tree was needed before an epizootic of Z. canadensis /SOPER and MACLEOD 1981/.
Specific behavioural modifications of infected insect may have an effect on dissemination
of pathogen. For instance, males of flies are significantly more attracted to Entomophthora
muscae-killed females than to uninfected females and so they can effectively transmit disease
within population /MØLLER 1993/. In many entomophthoralean pathogen-host systems
something like a negative geotaxis appears, when infected insects tend to move upwards just
44
before a death and die on the top of plants /BATKO 1974/. It leads to increase in the
probability for conidia to be discharged over a larger area. Similar phenomenon was also
observed for some Entomophthorales in aphid population /ROCKWOOD 1950, EVANS 1989,
JENSEN et al. 2001/. The position on the plant where infected aphids die depends in all
probability on the aphid species /ROY et al. 2002/. In laboratory experiments cadavers of S.
avenae were found higher on wheat than uninfected individuals, but this was not the case for
pea aphid cadavers /ROY 1997 in PELL et al. 2001, ROY et al. 2002/. Aphids killed by C.
obscurus were more often found on the lower parts of plants, but it was ascribed to more
favourable microhabitat for infection at a ground level /WILDING 1975, DEDRYVER 1981/.
Even, infected aphids were significantly more likely found on the undersides of leaves than
healthy aphids, what can be benefiting for the fungus because of better position for conidial
dissemination and protection from insolation /JENSEN et al. 2001/. Killed aphids were also
found more likely off the plants than were healthy aphids /JENSEN et al. 2001/. On the
contrary ROY et al. /1999/ noticed that infected aphids less likely dropped from a plant in
response to alarm pheromone, demonstrating a decrease in mobility. Infected aphids were
unable to respond to alarm pheromone, though they could still produce it. On the other hand,
A. pisum infected by P. neoaphidis demonstrated a behavioural response to the presence of
foraging coccinellids /Roy et al. 2002/. Despite the effect of entomophthoralean fungi on the
behaviour of their hosts is a characteristic property of these fungi this is still a part of
unexplored area especially in the aphid-pathogen systems. In certain aphid-pathogen systems,
e.g. C. coronatus-pea aphid system, a lack of effect on host’s behaviour is compensated for by
morphoanatomic and developmental adaptations of the fungus, e.g. polymorphic conidial
apparatus /BYFORD and WARD 1968, BATKO 1974/.
Fungal pathogens may be scattered within the host population or among the populations
not only by infected hosts themselves but also by parasites and predators active within the
host colonies. Parasites and predators of aphids come in contact with their prey and may
acquire inoculum from the environment and thus serve as passive vectors of disease among
populations /POPRAWSKI et al. 1992, ROY et al. 2001/ (see section 3.5.2.2.3).
3.5.2.2.2 Pathogen populations
Major biological properties of fungal pathogens involved in causing diseases are virulence,
pathogenicity, pathogen replication, pathogen population density, dispersal, spatial and
temporal distribution, as well as persistence in nature /TANADA and FUXA 1987, PELL et al.
2001/.
Infectivity, the ability of fungus to attack a host and cause infection, varies with different
fungal pathogens /PAPIEROK 1982/. A number of factors are involved in infectivity, including
the physiology of the host (e.g. defence mechanism), and the physiology of the fungus (e.g.
production of enzymes and toxins). Conidia of P. neoaphidis produced between the first and
the third hour of conidiogenesis are more infective than those produced later /LATTEUR et al.
1985/. Virulence, the ability to invade and injure the host’s tissues, is often measured by the
response of the host to a known inoculum dose, mostly a death from a median lethal
concentration LC 50 /PAPIEROK 1982/. Values of LC 50 range from few to hundreds of conidia
per mm2 depending upon fungal species and different isolates within species /e.g. PAPIEROK
and WILDING 1979, MILNER and SOPER 1981, OGER and LATTEUR 1985, FENG and JOHNSON
1991, XU and FENG 2000/. Strains of certain fungal species differ in their virulence. LATGÉ et
al. /1982, 1984b, 1986b/ reported 2 groups of C. obscurus strains with different
aggressiveness to pea aphid. Likewise, PAPIEROK and WILDING /1981/ demonstrated 2 types
of strain of C. obscurus differing in their physiological and infective properties and DUMAS
and PAPIEROK /1989/ isolated two strains of Z. radicans on the same day in the same locality,
which varied greatly in the virulence against adult mosquitoes. Insect pathogenic fungi may
produce toxins that enhance their pathogenicity /PAPIEROK and COREMANS-PELSENEER 1980,
45
for review see VEY et al. 2001/. A rapidly replicating pathogen is generally more virulent than
species that replicates slowly in the host, though other factors also may play an important role
/TANADA and FUXA 1987/.
A pathogen population density is one of the most important factors that determine if a
disease develops to epizootic. Most entomopathogens gain entry to the haemocoele by
penetrating the host’s cuticle using a combination of hydrolytic enzymes and mechanical
forces /BUTT et al. 1990, HAJEK and ST. LEGER 1994/. A speed of kill is influenced by a
number of infective propagules in contact with the host cuticle /BUTT and GOETTEL 2000/.
Pathogen population density relates to some characteristics inherent to pathogen, such as
reproductive rate and capacity to survive, and also some environmental factors, such wind and
rain /TANADA and FUXA 1987/ (see section 3.5.2.2.4). The time from the initial aphid contact
with conidium to death of the host and subsequent fungal sporulation is relatively short in
Entomophthorales. This can be as short as 3-6 days depending on fungus species, host, and
external conditions /e.g. WILDING 1969, DEAN and WILDING 1971, BROBYN and WILDING
1977, STEINKRAUS 1993a/. Generally, relatively fewer numbers of conidia are produced per
cadaver in Entomophthorales, when compared with, for example, Hyphomycetes. On the
other hand, fewer conidia are required for entomophthoralean infection /PELL et al. 2001/.
Thousands of conidia can be released from a single host, with numbers depending on cadaver
biomass and temperature /WILDING 1971, GLARE and MILNER 1991, DROMPH et al. 1997,
HEMMATI et al. 2001a/. About 3000 of primary conidia of N. fresenii are discharged per
infected cotton aphid and approximately ¾ of these conidia enter the air, while ¼ immediately
hit the leaf, on which the cadaver is located /STEINKRAUS et al. 1993/. Conversely, on average
50 000 to 60 000 conidia could be discharged from larger aphid species /WILDING 1971,
GLARE et al. 1986a, SIVČEV 1993/. In optimal conditions released conidia deposited on any
surface germinate within few hours to penetrate the host integument or to form secondary
conidia /WILDING 1971, BREY et al. 1986, STEINKRAUS et al. 1993/. At saturated humidity the
mean time for infection is 4.5 hours at 20°C /GLARE and MILNER 1991/. The pathogen density
or dosage can cause different types of disease that in turn could affect epizootiology, for
example, heavy doses can cause mortality more quickly than light ones. Rapid mortality, on
the other hand, reduces the replication of pathogen and the subsequent pathogen density when
the insect host dies /TANADA and FUXA 1987/. Little is known about a critical inoculum
threshold of pathogen population, at which an epizootic can develop in aphid populations.
Aerobiology and density of airborne conidia have been studied in naturally occurring
epizootics. During N. fresenii epizootics in the cotton aphid populations the number of conidia
present in the air is immense. Up to 90 000 of primary conidia per cubic metre of air were
collected above cotton fields and active discharge of conidia was observed mainly between
01:00 and 03:00 hr in the morning /STEINKRAUS et al. 1996b, 1999/. In the cereal aphid
populations the most number of P. neoaphidis primary conidia was recorded in the air over
fields between 04:00 and 08:00 hr in the morning when temperatures were between 10 and
16°C and humidity was greater than 90% /HEMMATI et al. 2001b/. These studies show that
conidiogenesis and sporulation take place when the external conditions, such as temperature,
humidity and light, are the most appropriate for the process. Air-dispersed conidia in the space
above crops demonstrated infectivity to aphids /STEINKRAUS et al. 1999/. Pathogen population
density in epizootics in many studies was chosen by multiple regression analyses as one of the
most important factor in fungal disease prevalence /e.g. WILDING 1975, SOPER and MACLEOD
1981, COREMANS-PELSENEER 1983, CAPPUCCINO 1988, STEINKRAUS et al. 1996b, 1999,
HEMMATI et al. 2001b /.
To effectively utilize entomopathogens as an agent for biocontrol of aphids, it is necessary
to also understand the biotic and abiotic conditions that facilitate dispersal and spatial
distribution of infection. Pathogenic fungi rely mainly on physical and biotic factors for their
46
dispersal and, in general, they have very limited capacity to disperse through their own
actions /TANADA and FUXA 1987/. However, the Entomophthorales possesses conidiophores
that forcibly discharge conidia by hydrostatic pressure /BEN-ZE’EV and KENNETH 1982a/ what
facilitate the inoculum spreading /PELL et al. 2001/. Values of maximum discharge distance of
primary conidia from aphid surface are generally between 6 and 9 mm /HEMMATI et al.
2001a/. Conidia of Entomophthora muscae (Cohn) Fresenius were even discharged up to 87
mm from killed fly and average discharge distance was positively related to cadaver size /SIX
and MULLENS 1996/. The conidia, when discharged into the air, are carried by wind currents,
which are the most important physical factor for dispersal /PELL et al. 2001/. Production of
replicate conidia may also increase the dispersal capacity /PELL et al. 2001/, however in some
genera, e.g. Zoophthora, Neozygites, certain types of higher-order conidia are not actively
discharged /BEN-ZE’EV and KENNETH 1982a, KELLER 1997/. Cadavers of aphids killed by
some species are fixed to plant surface what also enhance chances for dispersal of conidia
/PELL et al. 2001/. Some species released conidia with slime coats that serve in attachment to
insects as well as in dispersal /BREY et al. 1986, BOUCIAS and PENDLAND 1991/. Modified
behaviour of infected hosts may also contribute to the dispersal of pathogens (see section
3.5.2.2.1). There are studies on distribution of Entomophthorales in agroecosystems
/STEINKRAUS et al. 1996b, 1999, HEMMATI et al. 2001b/. Temporal distribution of infection
units in the host’s habitat is as important to the development of epizootics as a spatial
distribution. Susceptible host and infective pathogen must make contact in both space and
time for infection to occur /TANADA and FUXA 1987/. Numbers of conidia of certain
Entomophthorales discharged in the air have characteristic daily /WILDING 1970a, HARPER et
al. 1984, MILNER et al. 1984, STEINKRAUS et al. 1996b, 1999, HEMMATI et al. 2001b/ as well
as seasonal patterns /HARPER et al. 1984/. A diurnal periodicity in conidial production depends
on environmental conditions, mainly light and relative humidity. The fungi produce conidia
during the early morning hours when light conditions, humidity, and temperature are optimum
and at which time an inoculum transmission to susceptible hosts must occur, since after
daylight the conidia succumb to ultra-violet radiation and higher temperature /AOKI 1981,
HARPER et al. 1984, MILNER et al. 1984, STEINKRAUS et al. 1996b, 1999, HEMMATI et al.
2001b/. During winter and early spring months the environment is, theoretically, free of
infective inocula. The main source of the infective units are resting spores deposited in the
soil and/or on tree bark /BITTON et al. 1979, COREMANS-PELSENEER 1981, PERRY and LATGÉ
1982, HAJEK et al. 1998/. The resting spores have to germinate to produce germ conidia and
thus to provide an adequate density of inoculum, a fundamental prerequisite for disease
initiation in nature. Early spring temperatures induce asynchronous germination of resting
spores, which produce infective conidia over a period of several weeks. These spores infect
the first aphids occurring on the crop /PERRY and LATGÉ 1982/. The spores germinate under
certain environmental conditions after they have undergone a stage of dormancy /BITTON et
al. 1979, PERRY and LATGÉ 1982, BEN-ZE’EV et al. 1990/ and the period of dormancy is
influenced by temperature and humidity /PERRY and LATGÉ 1982/. For instance, 4-monthly
storage of C. obscurus resting spores at 4°C and 95% of relative humidity was necessary to
break their dormancy /LATGÉ et al. 1983/. In Israel it was observed that resting spores of N.
fresenii were synchronized to germinate in spring concurrently with a build-up of aphids on
citrus /BITTON et al. 1979, BEN-ZE’EV et al. 1990/.
Pathogens may persist and survive in abiotic environments (in stage of resting spores,
conidia) /e.g. BITTON et al. 1979, LATTEUR 1980, NIELSEN et al. 2003/ and biotic
environments of host’s habitat (in stage of hyphal bodies) /KELLER 1987b, FENG et al. 1992b,
NIELSEN et al. 2003/. Factors in the abiotic environment above the soil surface that most
significantly affect the persistence of fungi are sunlight (especially, ultraviolet light),
temperature and humidity. There is considerable information on the effect of these factors on
47
the pathogens. In general, the entomophthoralean conidia are relatively fragile and short-
lived, but can germinate quickly and produce replicate conidia thus prolong their persistence
/AOKI and TANADA 1974, PELL et al. 2001/. BROBYN et al. /1985/ demonstrated that survival
of P. neoaphidis primary conidia on leaves was greater when conidia were deposited on
underside (more then 7 days) than on the upper surfaces (up to 3 days) of leaves where the
fungus was not protected from sunlight. This is in agreement with results of FURLONG and
PELL /1997/ who also highlighted importance of higher moisture on the underside of leaves
for a longer persistence. In the field primary conidia of Z. radicans on foliage or soil lost their
viability within 24 hrs /FURLONG and PELL 1997/. Infectivity of C. obscurus conidia deposited
on surface of non-sterile soil persisted depending upon temperature from 2 days to 2 months.
The lower temperature of storage (within a range of 5 to 20°C) the higher persistence of
infectivity was recorded /LATTEUR 1980/. The author speculates whether conidia emitted in
the autumn could survive the winter and initiate life cycle in the spring. Conidia of C.
coronatus survived on cotton leaves under humid conditions for 70 days, but in glasshouse
conditions they remained viable just for 10-14 days /GINDIN and BEN-ZE’EV 1994/.
Capilliconidia are more resistant both to ultraviolet radiation /FURLONG and PELL 1997, UZIEL
and SHTIENBERG 1993/ and lower humidity /STEINKRAUS and SLAYMAKER 1994/ than conidia.
If abiotic conditions are not favourable for conidiogenesis, the aphid cadavers desiccate and
may temporarily persist in this anhydrobiosis to rehydrate and recover after a humid period
comes /GLARE and MILNER 1991, BEHRENS 1993/. The most number of resistant stages of
Entomophthorales, the resting spores, end up in the soil where they remain during the winter.
Soil is highly favourable environment for entomopathogen survival and is a natural reservoir
of entomophthoralean resting spores /COREMANS-PELSENEER 1981, LATGÉ and PAPIEROK
1988, NIELSEN et al. 2003/. Aphids and other insects can be infected with soil samples
collected in place where the mycosis have been active what validate theory of soil as a
pathogen reservoir /LATTEUR 1977, HAJEK et al. 2000, NIELSEN et al. 2001a, 2003,
MIETKIEWSKI and TKACZUK 2003/. The persistence of these spores in the soil has a long-term
significance for epizootics. Many resting spores do not germinate in the first year after
production and can remain viable in the soil for very long time /LATGÉ and PAPIEROK 1988,
WESELOH and ANDREADIS 2002/.
3.5.2.2.3 Transmission of infection
Transmission may be defined as a process, by which a pathogen or parasite is passed from
a source of infection to a new host. This process can be direct when fungus is transferred from
an infected host to a susceptible one without an intervention of any living agent, or indirect
when one or more species of intermediate hosts or vectors is involved. The most prevailing
transmission form among fungal pathogens is direct. A knowledge of transmission pathways
is fundamental to understanding disease dynamics and insect epizootiology /ANDREADIS
1987/. Transmission is normally divided into two categories according to the manner, in
which the pathogen is transferred within a host population. Transmission is horizontal when
the fungus is transferred from individual to individual by physical contact and it is vertical
when there is direct transfer of the pathogen from parent to its progeny /KŮDELA and POLÁK
1999/. Entomophthorales rely on horizontal routs of transmission thus the fungi are dependent
on a host density for their survival and dispersal /WILDING and PERRY 1980, PELL et al. 2001/.
Reliance on horizontal transmission means that the pathogen must either maintain itself
within the living host or cadaver throughout the year or produce a resistant stage that is
capable of surviving in the environment when susceptible stages of host are absent. In such
host-pathogen relationships disease prevalence is usually density dependent and increases
steadily or dramatically during a host population culmination /ANDREADIS 1987/. Vertical
mode of transmission is not expected to be in Entomophthorales parasiting aphids, since
48
embryos are not vulnerable to the infection thus a transovarial transmission is disabled /BUTT
et al. 1990/.
Dispersal or dissemination is defined as the capacity of a fungus to spread and distribute
itself within a host population and throughout the environment. This process within pathogen-
aphid system is partly discussed before from a viewpoint of host (see section 3.5.2.2.1) and
pathogen (see section 3.5.2.2.2). The successful long-term persistence of pathogens within a
host population is related to its ability to disperse. Pathogens with low dispersal capabilities
have a very low potential for developing epizootics even though they may be highly virulent
or survive effectively in the environment /TANADA 1963/. There are several ways, in which
entomophthoralean fungi are disseminated within host population or among host populations
in nature: by their own actions, by the behaviour and movements of infected hosts, by the
behaviour of non-host carriers, and by climatic and physical agents /e.g. ČUDARE 1990,
JENSEN et al. 2001, PELL et al. 2001, ROY et al. 2001/. The extent of entomophthoralean
dissemination depends presumably upon: (1) a density of host population, since the higher
density of host the higher probability of horizontal transmition by contact of healthy and
diseased aphids; (2) a concentration of fungal inocula in the environment; (3) a physiological
state of host organism; and (4) a properties of pathogen /ČUDARE 1990/. The forcible
discharge of conidia from infected insect is an important method of autodissemination among
entomophthoralean fungi /PELL et al. 2001/. Discharged conidia are anemochorous. They
enter the air current above crops with potential to be passively dispersed /HEMMATI et al.
2001b/. Distinctive movements or migration of diseased aphids also affects pathogen
dispersal within a host population /RABASSE and ROBERT 1975, WILDING and PERRY 1980,
ČUDARE 1990/. Positive correlation was found between the numbers of infected aphids and
live alate ones /CAGÁŇ and BARTA 2001/. Few studies considered whether entomophthoralean
species could move freely between different aphid species and spread thus fungal infection in
the agroecosystem /MILNER et al. 1983, STEENBERG and EILENBERG 1995/. Arthropod
enemies of aphids and entomopathogenic fungi co-exist in large aphid populations in the same
habitats. There are examples in the literature when hymenopteran parasitoids or predators
acted as passive vectors of entomophthoralean fungi to host populations during foraging
/POPRAWSKI et al. 1992, PELL et al. 1997a, ROY et al. 2001/, but there are also examples where
this did not occur /FURLONG and PELL 1996, FUENTES-CONTRERAS et al. 1998/. The extent to
which parasites and predators contribute to the dissemination of disease and development of
epizootics varies with each individual host-parasite-pathogen system. The production of
resting spores during unfavourable conditions ensures the survival of these fungi over periods
when no susceptible hosts are present and provides a mode of dispersal to new host
populations in subsequent years /PERRY and LATGÉ 1982/. Actions of climatic and physical
agents may also result in increased prevalence of disease within a host population by bringing
the pathogen into contact with new hosts /PELL et al. 2001/, or in a decreased prevalence of
disease by removal of inocula from the environment, which the host population inhabits /PELL
et al. 1997b/.
3.5.2.2.4 Environmental factors
Since Entomophthorales are transmitted horizontally in the environment, they are
depended considerably upon environmental conditions. Environmental factors may directly or
indirectly influence the trio of primary factors contributing to the epizootics of insect diseases:
a host population, a pathogen population, and a means of transmission. Environmental factors
do not act separately, but as a complex whole. Therefore, the analysis of single environmental
factor on epizootics is not always successful. Environmental factors, especially the climatic
factors, act on the capacity of the pathogens to survive in the biotope, a germination of the
pathogens, a conidial discharge as well as on their hosts /e.g. GLARE and MILNER 1991,
HEMMATI et al. 2001a/. Detrimental effect of high temperature, desiccation, and sunlight on
49
free pathogens is well known. Species with a resting spore stage in their life cycles are better
able to survive and persist in the environment /e.g. WILDING 1969, WILDING 1970b, BROBYN
et al. 1985/.
Temperature is one of the principal environmental factors influencing activity of
entomopathogens. Temperature is a factor, which effectively affects conidiogenesis,
germination of conidia, and incubation time /e.g. WILDING 1971, MILNER and BOURNE 1983,
MILNER and LUTTON 1983, GLARE and MILNER 1991/. Study of these effects is complicated
because the developmental rates of both pathogen and aphids are regulated by temperature. If
the temperature favours a quick generation time for aphids, but is above or below the
optimum for the pathogen, the insect population may still build-up to crop-damaging levels.
Conversely, if temperatures favour a brief incubation period for the pathogen, but retard insect
development, epizootics can result /BENZ 1987/. Many data on the influence of temperature
on diseases were collected under laboratory conditions. The mean optimum temperature for
fungi is normally between 20°C and 25°C with maximum of about 35°C and minimum of
about 5-10°C /ZIMMERMANN 1986/. The time to kill pea aphid by P. neoaphidis and C.
obscurus is dependent on temperature and infection does not occur at 0°C or at 30°C. The
time to kill varied from 5 to 16 days at 20 and 10°C, respectively /VORONINA 1968, WILDING
1970b/. The incubation period of diseases increases as temperature decreases within a certain
range of temperature /MILNER and BOURNE 1983/ and infectivity of fungi may also vary at
different temperature regimes /FENG et al. 1999, SHAH et al. 2002/. C. coronatus grows at
temperature of 16-30°C with optimum of 20°C /PAPIEROK 1985/, but strain isolated from
human mycosis had optimum at 37°C, which was lethal for the other strains /PAPIEROK et al.
1993/. Strains of C. coronatus isolated from soil in Israel had optimum temperature for spore
production at 20°C and for growth at 30°C /ALI-SHTAYEH et al. 2002/. Z. radicans in spotted
alfalfa aphid produced more conidia at 25°C than at 15, 20°C or 30°C /MILNER and LUTTON
1983/. Optimal temperatures for conidial germination are between 18-20°C and 25°C
/WILDING 1971, GLARE and MILNER 1991/. P. neoaphidis sporulated at temperatures from 11
to 25°C /SIVČEV 1993/ and not above 30°C /SIVČEV 1993, SIVČEV and MANOJLOVIĆ 1995/
what corresponds with results of SHAH et al. /2002/. The greatest number of conidia per
cadaver was released at changeable temperatures /SIVČEV 1993/. Temperature may influence
not only the number of conidia produced from aphid cadavers but also the maximum
discharge distance of conidia. Greater numbers of conidia are produced and they are shoot off
further at 18°C, lower and higher temperatures reduce these properties /GLARE and MILNER
1991, DROMPH et al. 1997, HEMMATI et al. 2001a/. Species of the family Neozygitaceae are
unusual in that they are common and more active in hot weather. The fact that Neozygitaceae
are adapted to hot and humid conditions has been reported by many researchers /GUSTAFSSON
1965, STEINKRAUS et al. 1991, KELLER 1997/. High temperatures that could occur in nature
only rarely have been used in laboratory tests. High temperatures may, for instance, occur on
the soil surface in summer /BENZ 1987/. Conditions of temperature in the top few centimetres
of soil may range over 40°C between daylight and night hours, and temperatures over 50°C
can occur /CARRUTHERS and SOPER 1987/. Unfortunately there are also only few data on the
heat resistance of fungi. KREJZOVÁ /1971b/ reported that hyphal bodies of C. obscurus lost
their viability at 40°C, resting spores do not germinate when exposed to temperature of 60°C
for 240min., or to temperature 100°C for 15 min. Resting spores of C. thromboides showed
somewhat higher resistance to these temperatures. Transmission of inoculum in the
environment may be influenced by temperature through sensitivity of discharged conidia to
higher temperature, desiccation or insolation /e.g. FURLONG and PELL 1997/.
Humidity is another important environmental factor affecting the course of epizootics in
insect populations. A water-saturated or near-saturated environment is essential for active
conidial discharge, germination, and infection initiation. According to MILLSTEIN et al. /1982,
50
1983/ a conidial discharge occurred when relative humidity in the crop exceeded 91%. This
result is in agreement with earlier observations that conidial discharge in two
entomophthoralean species, P. neoaphidis and C. obscurus, increased with increasing
humidity and that more conidia were produced from cadavers in contact with liquid water
than from those in a saturated atmosphere /WILDING 1969, 1971, VORONINA 1968/.
Sporulation was almost completely inhibited below 93% of relative humidity /WILDING 1969,
SIVČEV and MANOJLOVIĆ 1995/, but when the humidity rose again sporulation can
recommence /GLARE and MILNER 1991/. N. fresenii discharge most of its primary conidia at
night when the humidity is higher /STEINKRAUS et al. 1999/. Before daylight most of sensitive
N. fresenii primary conidia have germinated to form capilliconidia, which are more resistant
to lower humidity /STEINKRAUS and SLAYMAKER 1994/. ELLIOT et al. /2002/ studying
mycoses in mite populations pointed out that saturation deficits (high humidities) are mainly
determining a horizontal transmission, while the process of infection is not so affected by
abiotic conditions. Viability of primary spores of Z. radicans at different humidities was
studied and the best viability was recorded at humidity above 95% /GRIGGS et al. 1999/.
Influence of leaf wetness on infection of aphids was investigated by MILNER and BOURNE
/1983/. Establishment of epizootics in aphid populations are usually associated with periods of
rains or just after periods of rains. WILDING /1975/ reported that infections of the pea aphid
were positively correlated to the average rainfall recorded 12 days prior to disease
observations. Similar relationship was later observed by CAGÁŇ and BARTA /2001/. Others
have also tried to quantify this relationship. MISSONNIER et al. /1970/ determined that for an
enzootic of Entomophthora sp. to be sustained in an aphid population there must be a
minimum of 90% relative humidity for at least 8 hours per day. To increase disease levels to
epizootic proportions, the relative humidity must exceed 90% for 10 hours per day and there
must be 5 hours of rain per day for 3 consecutive days. Similar relationships were observed in
many studies on entomophthoralean epizootics /e.g. MACLEOD 1955, WILDING 1975,
ELKINTON et al. 1991, FENG et al. 1991, 1992a, CAGÁŇ and BARTA 2001/. VORONINA /1971/
used the hydrothermal coefficient (sum of precipitation x 10.0/sum of average temperature >
10°C) to define ecological zones in the former Soviet Union according to their ability to
support epizootics of Entomophthora sp. in the pea aphid populations. In those areas where
the coefficient exceeds 1.4, fungi regularly control pea aphid populations below economic
levels. When the coefficient is between 1.0 and 1.4, fungi are still important aphid mortality
factor, but some crop damage can occur. In very dry areas, where the coefficient is bellow 1.0,
the pea aphid causes crop losses almost every year. The attempt to predict areas where
epizootics will occur based on a measurement of moisture like that of Voronina was not
followed. Those predictions may have some worth, however, they cannot be treated as a rule.
KENNETH and OLMERT /1975/ reported collections of N. fresenii from very dry areas of Israel.
E. planchoniana, P. neoaphidis, and Z. radicans were recorded from two desert localities in
Mexico /SANCHEZ-PEÑA 2000/. Negative effect of rain on inoculum dispersal may also occur,
since rain may deplete the amount of fungal inocula in the vicinity of aphids by washing them
off plant surfaces /KISH and ALLEN 1978, FURLONG and PELL 1997/. Heavy rainfall lasting for
over 30 min. removes significant numbers of P. neoaphidis conidia particularly from the
upper surfaces of leaves. And 60 min. of heavy rainfall is necessary to remove aphid cadavers
from foliage. Conversely, light rain may compensate for the negative effects of heavy one by
enhancing sporulation and germination of spores /PELL et al. 1997b/.
Desiccation is one of decisive factors for the persistence of pathogens in nature. Generally,
fungal pathogens cannot survive desiccation and conidia are the most vulnerable units to
desiccation /PELL et al. 2001/. Exposure of N. fresenii primary conidia to 75% of relative
humidity for only 1 min. significantly reduced germination and subsequent formation of more
resistant capilliconidia. However, some capilliconidia remain infective for 2 weeks at 75% of
51
3. REVIEW OF LITERATURE: 3.6 Potential of Entomophthorales for biological control
relative humidity /STEINKRAUS and SLAYMAKER 1994/. Contrary to conidia, hyphae are more
resistant to desiccation. Aphid cadavers filled with hyphal bodies can be stored desiccated at
low temperature and so preserve fungal viability for several weeks /ROCKWOOD 1950,
WILDING 1973, LATTEUR et al. 1985/. Desiccated mummies of killed aphids can also preserve
fungal viability in nature. C. obscurus and P. neoaphidis infecting pea aphids were tolerant to
periods of low humidity and the fungi in aphid mummies could accumulate the effects of
short periods of high humidity for development of conidia /BEHRENS 1993/.
Positive stimulation of pathogens by light is reported rarely. EGE /1965/ confirmed a
stimulating action of the blue part of the light spectrum on certain Entomophthorales of
aphids. During conidiogenesis of P. neoaphidis, light increases the infectivity of conidia
/LATTEUR et al. 1985/. Under light conditions conidial rate of discharge reached higher values.
In dark it was maintained longer, whereas in light it soon slowed after reaching the maximum
/WILDING 1971/. The germination process of P. neoaphidis conidia was faster at light than in
dark /SIVČEV and MANOJLOVIĆ 1995/. These reports contrast to a number of reports of
negative effects of light especially on persistence of conidia in nature /e.g. ODUOR et al. 1996,
FURLONG and PELL 1997/. Ultraviolet radiation is by far the most important factor in conidia
mortality. Survival of the infective units is rapidly reduced after several minutes of exposure
to ultraviolet radiation /BROBYN et al. 1985, UZIEL and SHTIENBERG 1993, SIVČEV and
DRAGANIĆ 1994, FURLONG and PELL 1997/. Discharged spores were more persistent on lower
than upper surfaces of leaves due to a lower UV exposure on underside /BROBYN et al. 1985,
FURLONG and PELL 1997/. Since unprotected protoplasm is easily destroyed by the impact of
solar energy, the action of light may be destructive. Therefore many organisms have evolved
light-absorbing protective pigments. For instance, resting spores of N. fresenii have black
episporium, which shields the spore from penetration of light /BEN-ZE’EV et al. 1990/.
Pesticides are anthropogenic factors that are often in the environment of insect and their
pathogens, so they should be regarded as epizootiologically relevant factors. Much work have
been done to investigated the effect of different pesticides on some entomophthoralean fungi
and mycosis prevalence /e.g. SMITH and HARDEE 1996, LAGNAOUI and RADCLIFFE 1998,
HATTING et al. 1999b, MCLEOD and STEINKRAUS 1999, WELLS et al. 2000, LATTEUR and
JANSEN 2002/. Pesticides mostly affect conidial production and germination /WILDING 1982,
PERRY and LATGÉ 1983, KELLER 1986/. However, field effects of agrochemicals are probably
less severe than those in the laboratory. This is true especially for such pesticides, which have
a low persistence on a leaf surface. Therefore, in practice, the compatibility of
entomopathogenic fungi with agrochemicals is mainly influenced by the timing of application
and the coincidence of biological and chemical agents /ZIMMERMANN 1986/.
3.6 POTENTIAL OF ENTOMOPHTHORALES FOR BIOLOGICAL CONTROL

Although the Entomophthorales is important and worldwide-distributed group of insect
pathogenic fungi no species is currently produced commercially and augmented for aphid
control /HUMBER 1990, EILENBERG 2002/. The main problem is a production of infective and
stable biopreparation. These fungal entomopathogens do have, however, further weak spots
from a viewpoint of practical employ. For example, there are some disadvantages linked with
a short lifespan of infective inoculum in the environment what makes timing of inundative
applications difficult or impossible /LACEY et al. 2001/. Nevertheless, some attempts to
manipulate a disease timing in aphid populations in fields with overhead irrigation and
fungicide application have been made /MACLEOD and STEINKRAUS 1999/. The fungal control
agents are still believed to be effective and serve as alternatives to insecticides when fungal
strains are selected carefully. The most interesting strains for use in augmentation biological
control being those possessing a high infectivity, a high intensity of sporulation and a short
infection cycle /PAPIEROK 1982/. Strains selected for biocontrol should not have too narrow
52
specificity, being commercially advantageous if a product has a relatively wide host range
within an insect group containing several pest genera. However, the host range cannot be too
wide and obviously must exclude beneficial insects as well as other invertebrates and
vertebrates /SAMSON et al. 1988/. Successful utilisation of Entomophthorales requires first of
all increased pathogen virulence and speed of kill, then improved pathogen performance
under challenging environmental conditions, greater efficiency in their production,
improvements in formulation that enable ease of application, increased environmental
persistence, appropriate incorporation into integrated pest management, and acceptance by
growers and the general public /LACEY et al. 2001/. Apart from fungal properties mentioned
above other factors are necessary to consider with respect to classical or conservation
biological control /SHAH and PELL 2003, SHAH et al. 2004/.
3.6.1 HISTORICAL
Invertebrate pathology is just a recently organised discipline, but literature on fungal
parasites of insects and mites is extensive and long established. In modern history the
entomopathogenic fungi are known to be significant under natural conditions for over 150
years. However, insect diseases were probably first observed by sericulturists in the Orient
and early Japanese notes (about 900 AD) say about muscardine silkworms being used for the
treatment of palsy or paralysis /STEINHAUS 1956, 1975/. One of the earliest references to the
parasitism of insects by entomopathogens in modern times is that of DeGeer, who found
parasitic infection on flies in 1779 /ROBERTS and HUMBER 1981/. After nearly 60 years, in
1834, Italian Agostino Bassi, the pioneer laying the foundations for the study of infectious
diseases, elucidated the fungal nature of the white muscardine disease of silkworms /HALL
and PAPIEROK 1982/. In 1874 Louis Pasteur was the first who expressed the idea of using a
“mycelium” to control aphid populations /LATGÉ and PAPIEROK 1988/. Another eminent
scientist of that period who upheld insect pathology was Metchnikoff in Russia. He was the
first who mass-produced a fungus, Metarhizium anisopliae (Metchnikoff) Sorokin, for a
control of the wheat cockchafer and later the sugarbeet curculio /SAMSON et al. 1988/. A
common parasite of houseflies described in 1855 by Cohn and named as Empusa muscae,
subsequently renamed as Entomophthora by Fresenius, was the first description of
entomophthoralean species at all /BRADY 1981/. At the end of the 19th century much activity
directed towards the taxonomy of entomopathogenic fungi. Probably the most significant
taxonomic work on the Entomophthorales was that of Thaxter /THAXTER 1888/. Then a period
of intensive research on the use of entomopathogenic fungi in controlling agricultural pests
followed. Many attempts were made to use these beneficial organisms in the control of
various pest insects, but due to varying and often disappointing results the initial enthusiasm
decreased more and more. During the last 20 years, there has been a sort of renaissance in the
development of biological control methods and in the interest on fungal pathogens. This was
promoted by the increased use of pesticides and consequently by the finding of their negative
environmental impact.
Several researchers experimented with the use of entomophthoralean fungi as microbial
control agents in the late 19th century. The first documented case in which a member of the
Entomophthorales was exploited as a control agent dates back to 1895 in the South Africa
/SAMSON et al. 1988/. A fungus isolated from diseased locusts and believed to be a species of
the Entomophthorales was mass-produced for sale to farmers. Unfortunately, later, the
cultures were identified as Mucor sp. PETCH /1925/ deduced that the original field pathogen
was Entomophthora grylli Fresenius, but the initial isolations were contaminated and the
contaminant was distributed. And the reported field successes in fungal distribution were
probably due to natural epizootics of E. grylli. Cook /COOK 1892 in SAMSON et al. 1988/
remarked on the susceptibility of aphids to Entomophthora infections. Attempts to introduce
53
of inoculum of Entomophthorales to pest populations were conducted in North America.

Inoculum of Erynia radicans was successfully released against the European apple sucker in
apple orchards in Canada /DUSTAN 1924 in SAMSON et al. 1988/. Comparable trials followed,
among which can be mentioned those involving aphids: Myzus persicae on potatoes /HARRIS
1948/ and the spotted alfalfa aphid /HALL and DUNN 1958/. During last 3 decades many field
or laboratory trials to control aphids using entomophthoralean fungi with different results
were published in literature. In most cases aphid populations were not reduced sufficiently to
prevent crop damage suggesting that the fungi were not acting quickly enough /DEDRYVER
1979, WILDING 1981, LATGÉ et al. 1983, LATTEUR and GODEFROID 1983, WILDING et al.
1986a,b, 1990, ČUDARE 1990, SILVIE et al. 1990, SHAH et al. 2000a/.
3.6.2 STRATEGIES FOR BIOLOGICAL CONTROL

In general, biological control (biocontrol) of pests includes the use of living organisms
toward a reduction of pest population. As shown in Figure 4, the biological control is kept in
the framework of integrated pest management (IPM) as defined by KOGAN /1998/: “IPM is a
decision support system for the selection and use of pest control tactics, singly or
harmoniously co-ordinated into a management strategy, based on producers, society and the
environment.” Biological control can be defined as “The use of living organisms to suppress
the population density or impact of a specific pest organism, making it less abundant or less
damaging than it would otherwise be” /EILENBERG et al. 2001/. This definition stresses the
point that “living organisms” are used. This should include insect viruses as well, whereas
genes or gene fragments, metabolites from organisms (without the individuals producing
them) are excluded.
There are four different strategies for biological control (Figure 4): Classical biological
control, Inoculation biological control, Inundation biological control and Conservation
biological control /EILENBERG et al. 2001/. Inoculation biological control and inundation
biological control are often called augmentation /PELL at al. 2001/.
Figure 4. The relationship between biological control and other strategies for integrated pest
management /EILENBERG et al. 2001/
Integrated pest management
Mechanical, Host Biological Autocidal Biorational Conventional

physical and resistance control control chemical agents pesticides
cultural control incl. transgenic plants / biochemicals
Conservation Classical Inoculation Inundation

biological biological biological biological
control control control control
3.6.2.1 Classical biological control

The objective of classical biological control is permanent establishment of a biological
control agent for self-sustained long-term control. Classical biological control depends on
finding and releasing an appropriate biological control agent that is not native to the area
where the pest needs to be controlled. Public concern over negative side-effects on non-target
organisms after releasing and expected proliferation of biological agent may be an obstacle
/HAJEK and ST. LEGER 1994, EILENBERG et al. 2001, SHAH and PELL 2003/. The method can
54
be defined: “The intentional introduction of an exotic, usually co-evolved, biological control

agent for permanent establishment and long-term pest control” /EILENBERG et al. 2001/.
Several examples can be described where exotic entomophthoralean species have been
introduced for establishment against aphid pest. One of the examples is the release of an
Israeli isolate of Z. radicans to control the spotted alfalfa aphid, Therioaphis trifolii (Monell),
in Australia. The aphid caused serious damage to Australian alfalfa after its discovery in 1977
/MILNER and SOPER 1981/. Since surveys for native fungal pathogens of this aphid pest found
no candidates, it was concluded that exotic Entomophthora species should be evaluated for its
control /MILNER et al. 1980/. After the successful selection of the most promising Z. radicans
strain (from an extremely hot and dry Israeli site), it was released into field populations of the
spotted alfalfa aphid. The fungus dispersed rapidly into the fields where the releases were
made and was later found several hundred kilometres away /MILNER et al. 1982, MILNER and
MAHON 1985/.
Another example of a pathogen establishment in a new environment was the introduction
of N. fresenii into the cotton aphid populations in the San Joaquin Valley of California in
1994 and 1995 /STEINKRAUS and ROSENHEIM 1995, STEINKRAUS et al. 1998a/. Cotton aphids
in California lacked fungal pathogens. Summer releases of isolates from Arkansas, using
dried infected aphids, were moderately successful, resulting in the spread of the pathogen
from the release sites. Fungus activity continued until September or October, but epizootics
did not developed. Whether the pathogen persisted in California and caused long-term
suppression of the cotton aphid populations was not discussed. However, it is expected that
low relative humidity in the San Joaquin Valley may seriously limit the survival and spread of
the fungus /PELL et al. 2001/.
EILENBERG /2002/ presents his opinion about introducing Danish strains of P. neoaphidis
virulent to the green spruce aphid, Elatobium abietinum Walker, colonising spruce plantations
in Iceland. This pathogen has never been observed on this aphid species in nature in Iceland
/AUSTARÅ et al. 1997, NIELSEN et al. 2001b/. In the Western Iceland the only fungus
prevalent on this aphid was E. planchoniana, while in the Southeast Iceland only N. fresenii
was prevalent. This geographical distribution correlates with the distribution of two different
populations of E. abietinum found in Iceland. Both species established epizootics within their
distribution area /NIELSEN et al. 2001b/. The idea to artificially release E. planchoniana and
N. fresenii in Southeast or Western Iceland, respectively, in order to test whether they can
establish, proliferate and regulate the aphid populations was given as well /EILENBERG 2002/.
Besides efforts to introduce entomophthoralean fungi to aphid populations, other insect-
fungus-crop systems have been evaluated or tested for classical biological control. For
example, a Japanese strain of Entomophaga maimaiga was introduced to forests in the USA
against Lymantria dispar (Linné) /HAJEK et al. 1995, PELL et al. 2001/, or a Brazilian strain of
Neozygites floridana Fischer was introduced to cassava crops in Africa against
Mononychellus tanajore (Bondar) /ELLIOT et al. 2000, HOUNTONDJI et al. in EILENBERG
2002/.
3.6.2.2 Inoculation biological control

The common feature of inoculation biological control is that low levels of material are
usually released. The inoculative releases are carried out early in the season and it is expected
that inocula will reproduce, spread in the environment and control host population for an
extended period /PELL et al. 2001, SHAH and PELL 2003/. Success of the method depends on
the ability of the released organisms to multiply and then reduce the target pest population.
No permanent establishment of the biological control organisms is achieved /EILENBERG et al.
2001/. A definition of this method thus can be: “The intentional release of a living organism
as a biological control agent with the expectation that it will multiply and control the pest for
55
an extended period, but not permanently” /EILENBERG et al. 2001/. Due to the ability to
establish epizootics quickly in target pest populations, narrow host range, and potential to
persist in target insect populations, the entomophthoralean fungi fit very well in inoculation
biological control. There are several examples where natural levels of entomophthoralean
fungi are supplemented to effect aphid control in this way.
The work of HALL and DUNN /1957, 1958/ was the first major attempt to manipulate a
pathogen of the Entomophthora group for microbial control of aphids. In California three
entomopathogenic species (E. coronata, E. virulenta, and E. exitialis) were successfully
established in Therioaphis maculata populations on alfalfa by placing of fungal cultures
directly in the fields. DEDRYVER /1979/ dispersed local isolates of N. fresenii in living A.
fabae. The infected, still living aphids were then released to colonies of the aphid on bean
plants in glasshouse maintained under a regime of controlled relative humidity. The colony
growth was arrested effectively with the pathogen. Between 80 and 90% infection in treated
aphids was observed after 3 to 4 weeks. In England WILDING /1981/ released local strains of
P. neoaphidis and N. fresenii in aphid cadavers into A. fabae populations under field
conditions. During warm and dry seasons (in two years) the Entomophthorales introduced into
populations established briefly, but failed to spread. However, in cool and moist seasons
(other two years of experiments) the fungi spread rapidly and the yield of beans in treated
plots was higher that in untreated ones. In similar experiments WILDING et al. /1986b/
introduced P. neoaphidis into a field population of A. fabae using a three different type of
inocula, living laboratory-infected aphids, triturated cadavers of fungus-killed aphids, and a
homogenate of the fungus grown on agar plates. The fungus applied as triturate cadavers
established infection in the host population as effectively as by distributing laboratory-
infected aphids. More than 70% infection was recorded but the fungus failed to multiply fast
enough to protect the crop adequately. The application of homogenate even failed to establish
infection. LATTEUR and GODEFROID /1983/ introduced mycelium of P. neoaphidis (produced
in vitro) to populations of cereal aphids at fields in France. Although significant quantities of
produced conidia and favourable weather conditions were recorded, no positive results were
achieved. LATGÉ et al. /1982/ and SILVIE et al. /1990/ used fresh or dry mycelium of P.
neoaphidis for aqueous spray of aphid-infested plants in glasshouse. Although the fungus
sporulated and was able to infect some aphids, population suppression through horizontal
transmission was not achieved. Two Conidiobolus species, C. obscurus and C. thromboides,
were studied extensively for use in azygospore-based mycoinsecticides against Myzus
persicae on potatoes in the USA. These efforts were abandoned as technically impractical
/HUMBER 1990/. Classical introductions with fungus-mummified aphid cadavers as well as
inundative applications of marcescent mycelium of P. neoaphidis against cereal aphids were
carried out in England /WILDING et al. 1990/. Unfortunately, there were no significant effects
of treatment on the numbers of aphids. The artificial introduction of P. neoaphidis acted too
slowly and unpredictably. In a recent study by POPRAWSKI and WRAIGHT /1998/ small pieces
of sporulating P. neoaphidis mycelium were inserted into rolled wheat leaves with colonies of
Diuraphis noxia (Mordvilko) in Idaho (the USA). The fungus sporulated generously, 18% of
aphids were infected, and the fungus even spread rapidly to uninoculated nearby tillers, but
the fungus was slow to spread to the rest of the field.
3.6.2.3 Inundation biological control

Contrary to classical or inoculation biological control in inundation biological control the
control agent do not multiply or persist in the environment, but affects target pests
immediately after a mass-release. Generally, a large number of organisms is necessary to
achieve a sufficient effect /EILENBERG et al. 2001, SHAH and PELL 2003/. This can be
expressed as: “The use of living organisms to control pests when control is achieved
56
exclusively by the released organisms themselves” /EILENBERG et al. 2001/.

Entomophthoralean fungi possess some properties allowing them to use for inundation
biological control (e.g. high virulence), while other properties (e.g. short lifespan of conidia)
indicate that these fungi may fail as inundation biological control agents, especially in outdoor
environment. Indoor environment, like glasshouses, is the most promising site for this
strategy of biological control /EILENBERG 2002/. In many examples the trials are on a small
scale and are not repeated, making the differentiation between inoculation and inundation
biological control difficult. Generally, for Entomophthorales it is more usual that the intention
is to inoculate the crop with the fungus early in the season (what fully corresponds with
inoculation biological control) so that the fungus can establish and multiply, keeping aphid
populations below the damage threshold /PELL et al. 2001/.
Several aphid species were tested with different types of C. obscurus inoculum in
laboratory experiments /KREJZOVÁ 1972a/. The application of suspension of resting spores
produced in submerged culture to A. fabae on Sambucus nigra Linné resulted in 20%
mortality. The application of conidia by discharging them onto the aphids resulted in 40%
mortality in case of Megoura viciae Buckton on Vicia faba Linné, in 55% mortality in case of
A. fabae and in 100% mortality in a case of Aphis gossypii Glover. Another fungal species, C.
thromboides, C. coronatus, and C. destruens varied in percentage of mortality in laboratory
experiments from 28% to 88%. In the former Soviet Union ČUDARE /1990/ applied
suspension of fresh or dry culture (conidia or resting spores) of C. obscurus into populations
of more than 10 different aphid species with results of 71-99% mortality. On the base of
resting spores of a local strain of C. obscurus, a biopreparation “Entomoftorin” was prepared
in the former Soviet Union. The preparation could be stored at 4 °C for more than 8 years
without a loss of viability and virulence. After application usually 62-90% mortality of tested
aphids was recorded, but temperature of 20-24°C and relative humidity of 60-80% were
inevitable for the effectiveness /ČUDARE 1982/. In Russia resting spores of selected strain of
C. obscurus were exploited to develop biopreparation called “Mycoaphidin” with high
effectiveness for aphids /VORONINA 1997/. In Denmark EILENBERG /2002/ recommends
preparations of P. neoaphidis for inundation biological control of aphids in glasshouse crops.
The most recent attempts to control aphids in glasshouse were made by SHAH et al. /2000a/.
Experimental preparations based on alginate granules and mycelial mats of entomopathogenic
P. neoaphidis caused up to 14% infection rate in the potato aphid Macrosiphum euphorbiae
(Thomas) with foliar application or up to 36% infections with soil applications. Modes of
spray application of mycoinsecticides are discussed in detail by BATEMAN and CHAPPLE
/2001/.
It is obvious that large quantities of properly formulated inoculum are inevitable for use in
inundation biological control. As a matter of course not only the quantity but a the quality of
mass-produced inoculum is prerequisite /MAGAN 2001/. Therefore, in vitro cultivation is a
decisive step in the development of fungal biopreparations and the consecutive use of them in
biological control strategies. In vitro mass production of Entomophthorales can differ
significantly among genera or species, since nutritional requirements for growth are different
/LATGÉ 1981/. Conidiobolus grows quickly on standard media. Zoophthora, Pandora and
Batkoa are little more demanding for nutritional supplements. Entomophthora and
Entomophaga need more complex media /BAŁAZY 1993, LEITE et al. 2003/. Until now a
successful in vitro cultures of Neozygites have been reported only for species isolated from
mites and thrips /BUTT and HUMBER 1989, DELALIBERA 1996, KELLER 1997, GRUNDSCHOBER
et al. 1998, LEITE et al. 2003/. Neozygites species infecting aphids have not been isolated yet
in vitro /KELLER 1997/. At present the prospects of producing Neozygites species in vitro for
use as mycoinsecticides are uncertain. Out of the group of 31 aphidophagous
Entomophthorales described here, 20 species are known to grow and sporulate in vitro, 2
57
species can grow but not sporulate, 5 have not been isolated yet, and 4 species are
incompletely described or the isolation has not been tried.
Different types of fungal material have being tested for a mass production: conidia, hyphal
bodies, dried mycelium, or resting spores. Conidia of Entomophthorales, because of their
short lifespan, are of little value for biological control /COREMANS-PELSENEER 1981/. The
structures are also sticky, making them difficult to harvest from cultures and suspend
uniformly in water. In addition, if during formulation or application, the mucus surrounding
conidia is lost, a vital adhesion mechanism could be lacking /PELL et al. 2001/. Numerous
studies have already been focused to develop effective methods for mass production and they
have especially targeted at hyphal stages or resting spores. Hyphal bodies and resting spores,
contrary to conidia, are better type of inoculum, because they can be produced industrially
and may discharge infective conidia for some time after their application to crops /LATGÉ et
al. 1983/. When hyphal stages (hyphal bodies or dried mycelium) are applied into the fields,
the inoculum is expected to rehydrate and produce infective conidia in situ /PELL et al. 2001/.
In nature, mycelium of P. neoaphidis produced in vitro can project conidia for at most 3 days
after if has been sprayed onto plants /LATGÉ et al. 1983/. On the contrary, resting spores are
typical of asynchronous germination in field, the germination usually lasts one month and the
maximum daily level of germination never exceeds 10% of the applied spores. At 20°C the
resting spores produced in vitro stay viable for 1 to 2 months after application /LATGÉ et al.
1983/.
A mass production and drying method for Z. radicans, a “marcescence process” was
successfully developed /MCCABE and SOPER 1985 in PELL et al. 2001/. This technique
exploits the natural capacity of fungi to desiccate and rehydrate with resumption of their
normal development. The preparation was used in the field against Empoasca fabae and it
could be stored viable for at least 1 year in the freezer. Slightly modified marcescence process
of Z. radicans mycelium was developed to control Plutella xylostella Linné /LI et al. 1993/.
Research on the use of the most important and most frequent aphid pathogen, P. neoaphidis,
as an inoculative or inundative biocontrol agent is of limited success because of problems
with mass production, lack of stable inocula for testing, and dependence on suitable
environmental conditions for infection /MILNER 1997/. Some work has being done to develop
media and conditions for optimised mass production of P. neoaphidis. The pathogen do not
form resting spore stage in natural conditions and is thought to overwinter as desiccated
mummies filled with sclerotium-like masses of hyphal bodies /FENG et al. 1992b/. The fungal
mycelium can be mass-produced in 10 l batch fermenters in liquid media containing yeast
extract, glucose, and milk /LI et al. 1993/. Also, a semi-defined medium containing oleic acid
was developed /GRAY et al. 1990/, and a method for continuous P. neoaphidis culture was
designed /GRAY and MARKHAM 1997/. A maximum survival of mycelium produced in liquid
culture is 1 month at 4°C and 2 weeks at 20°C and non-swelling clay may improve this
storage up to 2 months at 4°C or nearly 1 month at 20°C /LATGÉ et al. 1983/. Aphid
pathogens in the genus Conidiobolus, primarily, were focused on resting spore mass-
production. Principally because of the simplicity, with which members of the genus produce
resting spores in vitro. SOPER et al. /1975/ produced C. thromboides resting spores using
media containing egg yolks, with yields approximating 2-3g of spores per egg yolk. The
mass-produced dry resting spores could be stored at 4°C for 1 year with no change in
germination. Liquid media were also used for in vitro production of resting spores. Semi-
defined media for resting spores production of C. thromboides /LATGÉ et al. 1977, 1978a,b/
and C. obscurus /LATGÉ 1980, LATGÉ and PERRY 1980, LATGÉ et al. 1983, REMAUDIÈRE
1983/ were proposed. To germinate the spores required overcoming of dormancy, a period of
about 3 months at 4°C and 95% relative humidity. Unfortunately, attempts to use in vitro-
58
produced resting spores of C. obscurus failed /WILDING et al. 1986a/ mainly due to a low or
asynchronous germination of spores.
Use of entomophthoralean fungi for biocontrol by dispersing sporulating cadavers or
moribund insects gave mixed results /e.g. WILDING 1981, WILDING et al. 1990/. Also spray
applications of unformulated hyphal or mycelial stage of P. neoaphidis did not provide
adequate control in glasshouse or field tests /LATTEUR and GODEFROID 1983, SILVIE et al.
1990/. Any inoculum produced in vitro have to be formulated for successful application,
stabilisation, storage, and improved efficacy in the fields /HALL and PAPIEROK 1982, SAMSON
et al. 1988, for review see WRAIGHT et al. 2001/. A new way of formulation of P. neoaphidis
mycelium have been tested recently /SHAH et al. 1998/. The hyphae of the pathogen were
immobilized in a matrix consisted of sodium alginate and calcium chloride was used as a
gelling agent. Conidia from freshly produced alginate beads caused 27 to 32% infection in pea
aphid in laboratory bioassays, what was not significantly different from infections in aphids
inoculated with fresh mycelial material. This type of formulation represents a promising
technique for practical application of fungal mycelium. Elevated humidity was recommended
for drying of P. neoaphidis mycelia at formulating and viability of preparation was best
maintained if stored at 10°C /SHAH et al. 2000b/. In order to enhance infectivity of alginate
granules to aphids, various additives were tested for their use during the formulation process
(e.g. sucrose, potato starch, or chitin) /SHAH et al. 1999/ and recent glasshouse trials using the
alginate granules have given promising results /SHAH et al. 2000a/. Soil applications were
more rapid in causing infections when compared with foliar applications. This was explained
by that the soil surface provided a buffered environment against changes in abiotic conditions
as well as a constant source of moisture for mycelial rehydration, unlike leaf surfaces where
P. neoaphidis was probably exposed to cyclical rehydration and desiccation. The production
of inocula of good quality in sufficient quantities for commercial applications and their
formulation require further research in order to define the media requirements during
production and to devise optimal formulating methods. However, the mass production and
formulation themselves are just single steps among a wide range of research activities, which
have to be done in the development of commercial mycoinsecticides /ZIMMERMANN 1986/.
Recently, several reviews have been presented on the progress in the field of fungal
biotechnology, including answers to questions about a selection of fungal organisms and
fungal strains for biological control, a choice of fungal propagule for the mass production,
factors governing in vitro growth and sporulation, and finally a mass production on either
liquid or solid media as well as a production strategy, a formulation, and a storage of
industrial preparations /SAMSON et al. 1988, BATEMAN and CHAPPLE 2001, WRAIGHT et al.
2001/.
Any fungus used for the biological control of insects may meet a diverse range of
pesticides applied to control the whole range of other pests on the same sites. It should be
remembered that these compounds could negatively affect entomopathogenic fungi. A way of
the pesticide activity may be different. Besides the direct toxicity for the fungi, the activity or
even the survival in the environment can be threatened by decreasing or eradicating their
natural hosts /HUMBER 1990/. Several studies have treated the sensitivities of
entomopathogenic fungi to a variety of pesticide groups and results have been different.
Fungicides may reduce prevalence of entomophthoralean pathogens in host populations
/ÖNCÜER and LATTEUR 1979, PICKERING et al. 1989b, SMITH and HARDEE 1996/, or even
delay an initiation of epizootics /WELLS et al. 2000/. Pesticides may affect germination of
conidia or inhibit the growth of hyphae /YENDOL 1968, LAGNAOUI and RADCLIFFE 1998,
HATTING et al. 1999b/. Entomopathogens showed their susceptibility to benomyl, tridemorph,
maneb, chlorthalonil, binapacryl, bitertanol, iprodione, mancozeb, captan, zineb, carboxin,
etridiazole, triphenyltin hydroxide, dithiocarbamate, and metalaxyl /e.g. YENDOL 1968, FRITZ
59
1976, ÖNCÜER and LATTEUR 1979, BRANDENBURG and KENNEDY 1983, CARRUTHERS et al.
1985, 1985, PICKERING et al. 1989b, SMITH and HARDEE 1996, LAGNAOUI and RADCLIFFE
1998, HATTING et al. 1999b, MCLEOD and STEINKRAUS 1999, WELLS et al. 2000, LATTEUR
and JANSEN 2002/.
3.6.2.4 Conservation biological control

Conservation biological control is distinguished from the other strategies in that any
natural enemies are not released. The principal object of the strategy is to keep pests under
economic threshold through protection and enhancement of biological control agents living
naturally in the environment. Conservation practices include limited and selective use of
pesticides but also active processes such as providing refuges adjacent to crops or within
crops, facilitating transfer of natural enemies between crops or directly provisioning food or
shelter for natural enemies /VAN DRIESCHE and BELLOWS 1996/. The method can be defined
as: “Modification of the environment or existing practices to protect and enhance specific
natural enemies or other organisms to reduce the effect of pest” /EILENBERG et al. 2001/. The
conservation biological control, sometimes also called a habitat manipulation, searches for
effective indigenous natural enemies and applies management practices that conserve and
promote them in the field. In annual crops, conservation approaches have potential,
particularly by the provision of permanent or semi-permanent refuges in the agroecosystem,
i.e. providing an environment similar to that in perennial crops /PELL et al. 2001, SHAH and
PELL 2003/. Several examples say about attempts to conserve the Entomophthorales in
agroecosystem.
In annual cropping systems a particular attention is given to a habitat manipulation in order
to provide permanent or semi-permanent reservoirs of entomophthoralean fungi - alternative
hosts for entomophthoralean fungi in agroecosystem, with a specific purpose of assisting
persistence and early-season multiplication of the fungi. These reservoirs could be managed
by field margin strips adjacent to hedgerows /ROY and PELL 2000/. Entomophthoralean fungi,
to be encouraged effectively by habitat manipulation, must be able to replicate extensively,
must persist for long periods of time in the reservoirs and must be able to disperse from the
reservoirs into pest hosts on crops /PELL et al. 2001/. Several aphid-pathogenic species are
known to overwinter in (alternative) hosts in hedges and forest borders /KELLER 1987b, 1998,
NIELSEN at al. 2001a/. Arthropod natural enemies conserved in the same sites could improve
the fungal chances to be transmitted locally or to the pest populations in adjacent crops /ROY
and PELL 2000/. The interactions between entomophthoralean fungi and arthropod natural
enemies in the environment, may be positive as mentioned above /FURLONG and PELL 1996,
2000, PELL et al. 1997a, ROY et al. 1998, ROY and PELL 2000, ROY et al. 2001/, or
antagonistic /POWEL et al. 1986b, BROBYN et al. 1988, PELL et al. 1997a, FURLONG and PELL
2000/. In Switzerland, it was observed that economically unimportant aphid species
developing in meadows in spring were important for the propagation of entomophthoralean
fungi in fields /KELLER and SUTER 1980/. If aphid populations in meadows were high in the
spring, P. neoaphidis and C. obscurus built up to sufficient levels to regulate aphid
populations in adjacent fields. Conversely, when the spring populations of aphids were scarce,
the entomophthoralean infections were low, as well. POWEL et al. /1986a/ found that
entomophthoralean fungi were more common at weedy edges of fields, and they supposed
that it was either because of higher humidity in weedy plots, or because of the spread of
infection from weed aphid species, or combination of both.
Modifications of the existing practice are often proposed as means to promote
entomophthoralean infections; for example less spraying with chemical pesticides or
irrigation are at least theoretically supportive to these fungi /EILENBERG 2002/. Irrigation has
been documented in several systems to artificially encourage epizootic development through
60
high relative humidity for sporulation and infection /WILDING et al. 1986b, PICKERING et al.
1989a, MCLEOD and STEINKRAUS 1999/. However, an excess of precipitation (irrigation) may
be undesirable by lowering a density of airborne conidia and washing spores off the aphid
cadavers what may consequently decrease an infection potential of the Entomophthorales in
the area /KISH and ALLEN 1978/.
As described above intentional modifications in the conventional practice and
environmental manipulation may be supportive to development and distribution of the
entomophthoralean fungi within agroecosystem. A simple reliance on the natural occurrence
of the entomopathogens is risky predominantly due to the unpredictability of factors that
govern epizootics. However, there is an example of utilization of natural epizootics in
regulation of the cotton aphid, A. gossypii, a significant secondary pest of cotton in Arkansas
(the USA). The entomophthoralean fungus N. fresenii often reduces the requirement for
chemical control of the pest /STEINKRAUS et al. 1991, 1995/ and the N. fresenii epizootics can
be predicted at least 1 week in advance by careful pest monitoring and diagnosis of aphid
samples /HOLLINGSWORTH et al. 1995/. If fungal prevalence is 15% or higher, there is a strong
likelihood that the aphid population will decline within a week, due to occurrence of
epizootic. If it is 50%, there is a strong likelihood that the aphid population will decline within
a few days. When disease prevalence exceeds 15%, growers are advised to refrain from
insecticide application, thus saving money, preserving beneficial insects and reducing
environmental contamination by pesticides /STEINKRAUS et al. 1996a, 1998a, STEINKRAUS
and BOYS 1997/.
3.6.3 BIOCHEMICALS AND ENTOMOPHTHORALES

Parasitic fungi are known to produce metabolites toxic to insects and, therefore, are
attracting attention as potential biological agents of insect pests /CLARKSON and CHARNLEY
1996/. In Hyphomycetes several toxic compounds have been recognized and characterized as
potent but not specific entomopathogens /KHACHATOURIANS 1996/. Entomophthorales are
known as species-specific entomopathogens with a rather narrow host range /PELL et al.
2001/. Recently a proteinaceous mycotoxin 30 kDa have been documented for C. coronatus,
isolated and characterised /BOGUŚ and SCHELLER 2002/. This success has given a hope for a
future use of entomophthoralean secondary metabolites as biorational chemical agents with
rather high specificity. In Russia even two mycotoxins isolated from C. obscurus have been
utilized to develop biopreparations called “Mycoaphidin T” and “Entoks” /VORONINA 1997/.
The “Mycoaphidin” is characterized with high effectiveness; 70-100% mortality was recorded
after 1% concentration of the biopreparation had been applied to various aphid species
populations. Lower mortality (50-60%) was observed only for M. persicae.
61
4. MATERIALS AND METHODS: 4.1 Survey of Entomophthorales in aphid populations
4. MATERIALS AND METHODS

4.1 SURVEY OF ENTOMOPHTHORALES IN APHID POPULATIONS
From 1999 through 2002 numerous localities were visited throughout Slovakia during
vegetation periods and colonies of various aphid species were searched in order to determine
a spectrum of entomophthoralean fungi parasitic to aphids. The observations started at the
beginning of March and usually finished at the end of November depending on weather
conditions and development of aphid populations. Altogether 70 different localities were
irregularly visited during the surveys and different types of sites were visited. The visited sites
varied for sampling occasions and the survey was mainly focused on aphids infesting
agricultural and horticulture crops, but observations were extended to include non-production
sites, lowland meadows, forests, hilly meadows, and vegetation growing by fields. The
characteristics of the surveyed localities are in the Table 1.
A similar survey of parasitic mycoflora in aphid populations was also conducted in some
parts of Austria in 2000 and 2001. Aphids were collected at 19 different localities in the
country; Vienna (10 sites), Lower Austria (6 sites), Upper Austria (1 site) and Burgenland (1
site). Sampling was performed during one period in 2000: in October (6.-31.), and during five
periods in 2001: in June (6. and 27.-28.), July (10.-13.), September (20.-25.), and in
November (8.-11.). The study was conducted through cooperation with the Institute of Forest
Entomology, Forest Pathology and Forest Protection at University of Natural Resources and
Applied Life Sciences in Vienna, and supported by the University Jubilee Foundation of the
City of Vienna (Grant H-78/2000).
Table 1. Characteristics of localities visited in Slovakia during 1999-2002

Locality Landform types* Altitude /m/ Climatic region
and subregion **
Bajč plain not dissected 121 T1
Bátorove Kosihy hill land moderately dissected 123 T1
Belá hill land medium dissected 456 M7
Bešeňov plain not dissected 121 T1
Bíňa plain horizontally dissected 132 T1
Bobrovník upland medium dissected 568 C1
Chľaba plain horizontally dissected 117 T2
Čavoj lower highland very strongly 534 C1
dissected
Černík plain not dissected 129 T1
Demandice plain not dissected 143 T2
Devičie hill land strongly dissected 277 T5
Dolná Malanta hill land moderately dissected 173 T4
Dolné Krškany plain horizontally and vertically 135 T2
dissected
Dolný Jatov plain horizontally and vertically 116 T2
dissected
Dolný Kubín hill land medium dissected 468 C1
Dubno hill land strongly dissected 234 T7
Fačkov hill land medium dissected 536 M7
Gbelce hill land moderately dissected 144 T1
Habovka lower highland medium 730 C2
dissected
62
Table 1 continues
Harmanec lower highland very strongly 440 M7
dissected
Huncovce hill land moderately dissected 639 C1
Ivanka pri Nitre hill land medium dissected 146 T2
Kamenica nad Hronom hill land moderately dissected 117 T1
Kláštor pod Znievom hill land moderately dissected 500 C1
Komjatice plain not dissected 130 T2
Komjatná 15 634 C1
Korytnica hill land strongly dissected 680 C1
Kostoľany pod Tribečom hill land moderately dissected 245 M3
Kunerad hill land medium dissected 494 C1
Lukáčovce hill land moderately dissected 183 T4
Malá nad Hronom hill land moderately dissected 140 T1
Malé Kosihy hill land medium dissected 115 T1
Malý Cetín plain horizontally and vertically 135 T2
dissected
Mojzesovo plain horizontally and vertically 130 T2
dissected
Motyčky lower highland very strongly 678 C1
dissected
Mudroňovo hill land moderately dissected 150 T1
Mužľa hill land medium dissected 121 T1
Námestovo hill land moderately dissected 614 C1
Nitra hill land medium dissected 173 T2
Ondrochov plain not dissected 125 T1
Oravský Podzámok hill land medium dissected 511 C1
Oravská Polhora hill land medium dissected 688 C1
Pastovce plain not dissected 124 T2
Plášťovce hill land medium dissected 155 T2
Pozba hill land moderately dissected 156 T1
Rabča upland strongly dissected 661 C1
Rastislavice plain horizontally and vertically 124 T2
dissected
Ružomberok upland medium dissected 494 M5
Ružový Dvor plain horizontally and vertically 130 T2
dissected
Selice plain not dissected 113 T2
Slaská hill land strongly dissected 426 M3
Sliač upland medium dissected 370 T7
Spišské Podhradie hill land moderately dissected 435 M2
Staré Hory lower highland very strongly 480 C1
dissected
Strážne plain not dissected 106 T3
Streda nad Bodrogom plain horizontally and vertically 105 T3
dissected
Šurany plain not dissected 123 T1
Trávnica hill land moderately dissected 130 T2
Uľany nad Žitavou plain horizontally and vertically 126 T1
dissected
63
Table 1 continues
Valča hill land medium dissected 450 M7
Veľká Lehota hill land medium dissected 576 M3
Veľké Bielice hill land moderately dissected 193 T4
Veľký Cetín plain horizontally and vertically 137 T2
dissected
Veľký Kýr plain not dissected 130 T2
Vlkas plain not dissected 136 T2
Vyšné Hágy hill land medium dissected 1066 C1
Zázrivá hill land strongly dissected 600 C1
plain horizontally and vertically 144 T2
Zbehy
dissected
Zliechov upland medium dissected 603 C1
Žemberovce hill land moderately dissected 226 T4
*landform types after /ABAFFY et al. 2002/

** Characteristics of regions and subregions /ABAFFY et al. 2002/
≥
warm region (T): 50 or more summer days annually in average (days with daily maximum air temperature
25°C)
subregion: T1 warm, very dry, with mild winter
T2 warm, dry, with mild winter
T3 warm, dry, with cool winter
T4 warm, moderately dry, with mild winter
T5 warm, moderately dry, with cool winter
T7 warm, moderately humid, with cool winter
moderately warm region (M): less then 50 summer days annually in average (days with daily maximum air
temperature ≥ 25°C), and July mean temperature 16°C or more
subregion: M2 moderately warm, moderately humid, with cold winter, valley/basin
M3 moderately warm, moderately humid, hilly land or highlands
M5 moderately warm, humid, with cool to cold winter, valley/basin
M7 moderately warm, very humid, highlands
cool region (C): the July mean temperature less than 16°C
subregion: C1 very humid, moderately cool, mean July temperature ≥ 12°C to < 16°C
C2 very humid, cool mountainous, mean July temperature ≥ 10°C to <12°C
4.1.1 COLLECTING AND HANDLING OF FUNGUS-KILLED APHIDS

All cadavers of aphids (fungus-killed aphids) were collected on a random basis. If plants
were found infested with aphids, the aphid colony was visually searched and cadavers were
collected. Recognition of fungus-killed aphids in colonies was made easier by characteristic
symptoms, by which diseases usually manifest themselves. Only cadavers with external
symptoms of entomophthoralean disease (e.g. a conspicuous position on the leaves, colour
changes, an adhering of cadavers to a surface of plants by rhizoids, a mycelial mat
overgrowing host body, a whitish halo of conidia deposited around the cadavers etc.) were
collected. The cadavers were carefully removed from the plant substrate with fine-tipped
forceps, or to avoid an aphid’s body destruction the cadavers were placed into a plastic box
with a piece of supporting substrate. As many as possible of aphid cadavers were transferred
into a plastic box from plants at each site. If fungus-killed aphids of certain host plant showed
the same external signs of disease, they were suspected being infected by the same pathogen.
Aphids like those were sampled in one plastic box. Aphids with apparently different
symptoms of disease were sampled individually.
Fungus-killed aphids collected at fields were used for pathogen identification as soon as
possible. And some part of the samples were deposited in herbarium prepared as air-dried
cadavers and stored at approximately +4°C in dark conditions.
At the time of collecting following data were recorded:
64
- locality
- date
- collection number
- name of the aphid host (genus, species)
- mane of the host plant
- prevalence of disease (epizootic, enzootic)
4.1.2 IDENTIFICATION OF ENTOMOPHTHORALEAN PATHOGENS

In laboratory the cadavers were subjected to a microscope examination to find
characteristic fungal structures and to confirm the infection. The cadavers were examined as
soon as possible after collection to avoid invasion of cadavers by fast-growing saprophytic
organisms complicating the true pathogen determination. Dimensions of hyphal bodies,
primary conidia, secondary conidia, conidiophores, resting spores and nuclei were measured.
Measurements of fungal structures were made from preparations mounted in aceto-orcein
/KELLER 1987a/ using a magnification of x720 (16 x 45). For each sample of material the
measurements were made on 25 objects per individual aphid, as 1 series. If possible, at least
four series were made. For each series a mean value of fungal structure dimension was
calculated. A final value of each dimension consists of two ranges: the range of the extreme
mean values of all the series and the range of extreme values of the measurement (in
parentheses).
To obtain a pure batch of primary conidia for the exact measurement, freshly killed aphids
were individually transferred from the plastic box to a piece of wet filter paper at a bottom of
a small Petri dish (a diameter of 50mm). After a sporulation had started a microscope slide
was put directly over the cadaver at a distance of 2-3mm and the dish was closed with the
Petri dish lid. In this simple humid chamber, primary conidia shooting out of conidiophores
were projected onto the slide /KELLER 1999/. The cadavers were left sporulating under the
slide only for 15-30 min. and the conidia were immediately mounted to prevent a
resporulation.
Secondary conidia were obtained from the primary ones deposited on the slide /KELLER
1999/. The slide with primary conidia was placed in a Petri dish on a wet filter paper. A
second slide was placed above the primary conidia and held in a distance of about 1-2mm.
Projected secondary conidia were collected on the upper slide and capilliconidia remained on
the lower slide.
Hyphal bodies were observed in infected still living aphids, which were also collected and
stored in 70% ethanol.
Sporulating fungal structures, rhizoids, pseudocystidia and resting spores were observed in
squash preparations when a portion of aphid cadaver was mounted on a slide in a drop of
aceto-orcein and gently pressed with a cover glass.
The keys by KELLER /1987, 1991/, BAŁAZY /1993/, and HUMBER /1997/ were used for the
identification.
4.1.3 COLLECTING AND HANDLING OF LIVING APHID SAMPLES

During the survey samples of living aphids were also collected from aphid colonies where
a disease was present. If possible adult alate or apterous individuals were only sampled. The
living aphids were stored in small polystyrene tubes (10 x 70mm) filled with 70% ethanol
/EASTOP and VAN EMDEN 1972/. The samples were used for an identification of aphid species
and a measurement of fungal structures developing inside host bodies at the beginning of
infection process (e.g. protoplasts, hyphal bodies).
65
4. MATERIALS AND METHODS: 4.2 In vitro isolation of entomopathogens and maintenance of cultures
4.1.4 IDENTIFICATION OF APHID SPECIES

Aphid species were identified using taxonomic features of alate and/or apterous adults
collected in the fields /HEIE 1980, MIYAZAKI 1987, BLACKMAN and EASTOP 1994, 2000/.
Clearing procedures for mounting the aphid samples on slides was performed after
HEIKINHEIMO /1988/ and EASTOP and VAN EMDEN /1972/. The keys by HEIE /1980, 1982,
1986, 1992, 1994, 1995/, BLACKMAN and EASTOP /1994, 2000/, and TAYLOR /1980/ were
used for the identification.
4.2 IN VITRO ISOLATION OF ENTOMOPATHOGENS AND MAINTENANCE OF CULTURES

4.2.1 PREPARATION OF CULTURE MEDIUM
Three types of culture media were used for isolation of entomophthoralean fungi or
maintenance of the isolates:
SDAYEM – Sabouraud dextrose agar (HiMedia®) (59g/l), dextrose (20g/l), agar (HiMedia®)
(20g/l), yeast extract (Imuna®) (10g/l), cow’s milk (150ml/l), and 6 egg yolks
EY – pure coagulated egg yolks
EYM – egg yolk (600ml/l), and cow’s milk (400ml/l)
SDAYEM /PAPIEROK and HAJEK 1997/:
The different ingredients of the medium were prepared separately as follows:
- for Sabouraud dextrose agar: exact amount of Sabouraud’s dextrose agar was put in a flask,
supplemented with a proportional volume of distilled water and sterilized at 120°C for 30
min.; the agar was then cooled down to 50°C;
- simultaneously a following mixture was prepared: exact amounts of dextrose, yeast extract,
and agar were placed in a flask, supplemented with a proportional volume of distilled water,
sterilized and cooled down as previous mixture;
- for milk: cow’s milk was poured into a beaker, the beaker with milk was tightly closed with
aluminium foil, sterilized at 120°C for 30 min., and kept at room temperature;
- for egg yolk: fresh hen’s eggs were placed in a mixture of 90% ethanol and 2% sodium
hypochlorite (200ml + 800ml) for surface sterilization; the more rounded pole of each egg
was sterilized in the flame for few seconds, the eggshell was carefully broken at the poles
with forceps, and the yolks were gently removed and poured into a sterile beaker;
- the sterilized milk was added to yolks in the sterile beaker, and the mixture was stirred with
a sterile glass rod until it was homogenous;
- the sterilized and cooled down Sabouraud’s dextrose agar and dextrose-yeast extract-agar
were added to egg yolk-milk mixture, stirred, and poured immediately into sterile Petri dishes
- the medium in dishes, after it hardened, could be stored in a refrigerator for several weeks
EYM /PAPIEROK and HAJEK 1997/:
Both ingredients were prepared separately and then mixed together in a sterile beaker. Milk
was sterilized in the same way as explained above for the previous medium, and egg yolks
were separated like was described above. Prepared medium was distributed into Petri dishes,
sterilized in the oven at 90°C for 45min., and cooled dishes were stored in a refrigerator.
EY /MÜLLER-KÖGLER 1959/:
Following the procedure with eggs like presented above, the egg yolks were separated
from egg whites, homogenized in a beaker, poured into small Petri dishes, and sterilized in the
oven at 90°C for 45°C.
66
4. MATERIALS AND METHODS: 4.3 Biological characteristics of Pandora neoaphidis isolates
4.2.2 ISOLATION OF PATHOGENS

Two methods and all the three types of media were
used to isolate the pathogens.
A. “Ascending conidia showering method”
In this method the isolation of fungi is direct
through conidia and can be utilized only for species
with actively discharged conidia. Original procedure
designed by KELLER /1999/ was modified.
Aphid cadavers bearing conidiophores were placed
on a wet piece of filter paper, which was attached to
the bottom of small Petri dish, to induce sporulation.
Sporulating cadavers were then put on a wet piece of
sterile cotton, which was attached to the inside of _______________________________
sterile Petri dish lid (Figure 5). Under sterile Figure 5. “Ascending conidia
conditions the lid with sporulating cadaver was showering method” for isolation of
inverted and covered with the Petri dish bottom Entomophthorales
containing a culture medium. So that the sporulating
cadaver was just 2-3mm below the surface of medium and projected conidia were collected
directly onto the medium surface. Conidia were collected for about 30 min. The longer period
of medium inoculation, the higher probability of contamination. After enough conidia were
collected, the lid bearing cadaver was replaced with another sterile lid and the Petri dish was
inverted bottom down.
B. “Whole cadaver method” /KELLER 1999/
This method involved a simply placing of cadavers on solid media. Prior to inoculation of
the medium with aphid cadavers, the aphids were surface sterilized. Fresh non-sporulating
cadavers were sterilized in 70% ethanol for about 10 sec., subsequently in 2% sodium
hypochlorite for 2 min., and washed in sterile water for 2 min. in 2-3 replications.
Incubation conditions and post-inoculation monitoring:
The culture media with conidia or aphid cadavers were incubated under temperature of 20
± 2°C and continuous illumination. The Petri dishes were daily observed in order to monitor a
germination of the conidia and a contamination with fast-growing saprophytes. Those plates
with contamination were discarded. After a week small colonies of entomophthoralean
mycelia could be observed onto the surface of medium plates.
4.2.3 MAINTENANCE OF CULTURES IN VITRO

The SDAYEM culture medium was only used for keeping Entomophthorales isolates in
culture. The cultures were maintained at 16-18 ± 2°C and a constant illumination. The
cultures were checked weekly and a frequency of subculture depends upon the fungus species.
However, transfers were usually made once a month at those conditions.
4.3 BIOLOGICAL CHARACTERISTICS OF PANDORA NEOAPHIDIS ISOLATES

15 isolates of Pandora neoaphidis (Table 25, see appendix), the most common
entomopathogen of aphids, isolated from various aphid species were involved in the
bioassays.
71
4.3.1 SIZE OF PRIMARY CONIDIA

A piece of sporulating fungal mat, taken from a fast growing margin of a culture, was
placed in the bottom of sterile Petri dish lined with water-soaked filter paper. Above the
mycelium a cover glass was fixed in a distance of 2-3mm. Conidia were collected on the
cover glass for 15 min. and conidial dimensions (length, diameter, and ratio of
length/diameter) were determined for 4 x 25 conidia for each isolate in a microscope (at
magnification of 16 x 45) fitted with a scaled eyepiece. For each replicate an average was
counted and obtained data were analysed by a one-way ANOVA (MS Excel 2000).
4.3.2 GERMINATION OF PRIMARY CONIDIA

Conidia for the experiment were obtained from the sporulating fungal mat as described
above. The conidia were collected on three substrates; a sterile cover glass, a square piece (10
x 10mm) of pure water agar (2%), and on a square piece of YG agar (2% yeast extract, 3%
glucose, 1.5% agar) /SIEROTZKI et al. 2000/. The conidia were incubated for 4 h at 100% and
75% of relative humidity, at 20 ± 2°C, and in the light. The humidity was controlled through a
method of saturated water solutions proposed by WINSTON and BATES /1960/. The
germination experiment was carried out at five replicates for each substrate and relative
humidity level. 5x20 conidia of each treatment were assessed for germination. The germ-tube
formation and secondary spore formation were recorded separately for each isolate.
4.3.3 RADIAL GROWTH RATE

SDAYEM medium was used to measure a radial growth rate of fungal colonies of the
isolates. 5-mm circular pieces cut off from margins of colony of tested culture were separately
transferred to the centre of culture medium in Petri dishes (70mm diameter) with five
replicates per each isolate. Subsequently, the Petri dishes were maintained at 20 ± 2°C under
constant illumination and examined with 5-day intervals for measurements of fungal colonies.
The experiment was finished on the 35th day, or when the dish was overgrown completely by
the mycelium. The daily rate of radial growth was calculated as x – 19.63 / t, where x is the
area of fungal colony (in mm2) measured at the time t expressed in days. The radial growth
rate was compared among the isolates by a one-way ANOVA (MS Excel 2000).
4.3.4 BIOMASS PRODUCTION OF THE ISOLATES

Fungal biomass was grown in a liquid medium /SIEROTZKI et al. 2000/ containing 1% yeast
extract, 10% milk, and 1.6% glucose. Before the biomass production experiment was
established a preculture had to be grown. 5 pieces of fast growing margins of culture were
added to 25ml of medium in a 100ml flask. The flasks were incubated at 20 ± 2°C in the dark
and agitated in shaking machine for 5 days. To establish a production culture 7ml of the
preculture was added to 40ml of medium in a 100ml flask. The cultures were incubated under
the same conditions as the precultures for 5 days. The biomass was harvested by filtration and
the mycelium was desiccated at 50°C for 60 min. on a filter paper. Dry mass of the mycelium
was weighed. The experiment was carried out at 6 replicates for each isolate and the means of
harvested biomass were compared among the isolates by a one-way ANOVA (MS Excel
2000).
4.3.5 VIRULENCE OF THE ISOLATES

The bioassay for assessment of virulence of the isolates implies following steps:
4.3.5.1 Obtaining of sufficient amount of infective inoculum
72
Pure cultures of entomopathogenic fungi were obtained by isolating of pathogens directly

from dead aphids naturally infected at fields (see section 4.2.2).
Production of inocula was performed on the same type of medium (SDAYEM), on which
the cultures were isolated and maintained. Since the fungus grew on the medium rather slow,
agar plates (50 mm Petri dishes in diameter) were inoculated with 3 pieces of a mother fungal
mat by evenly distributing them onto each agar plate in order to prepare a sporulating culture
in relatively short time. The inoculated plates were incubated at 20 ± 2°C with constant
illumination. Under these conditions the plates were after 10-15 days completely covered with
sporulating mycelium and ready for use in the bioassay.
4.3.5.2 Obtaining of disease-free aphids

The pea aphid, Acyrthosiphon pisum Harris, involved in the bioassays, was obtained from
their natural habitat, a pea crop at the locality of Dolná Malanta. One aphid colony was
transferred to the laboratory in a plastic box with a part of host plant and carefully examined
to confirm the absence of natural enemies (pathogens, parasitoids, or predators). Single vital
apterous adult was placed individually in plastic box supplemented with food sources. To
obtain disease-free aphids, the adult was left in the rearing plastic box for 2 days until it
produced the young. After 2 days the mother aphid was removed and its progeny were
supplied with a fresh food. They were kept at temperature of 20 ± 2°C and a photoperiod of
16 hr in the rearing box for 1 week. The small colony, a clone of one mother, was utilised to
establish a single strain culture of aphid.
Rearing of pea aphid was carried out on potted pea plants in controlled environment room
maintained at 20 ± 2°C, 40-60% of relative humidity and under a photoperiod of LD 16:8 h
with a sufficient intensity of light to ensure a rapid plant growth. The potted pea plants were
placed inside rearing cages. The cages consisted of a solid wood frame covered with fine
nylon mesh on every side. But the top of cage was covered with a glass plate for easier
observation and to give the plants maximum light. The aphids could be easily reared in such
cages and fresh host plants were needed to be supplemented weekly /ADAMS and VAN EMDEN
1972, FORBES et al. 1985/.
4.3.5.3 Exposing of tested aphids to various quantities of inoculum

Tested aphids were exposed to inoculum by method of placing the insects under
sporulating
culture of
pathogen /LATGÉ
and PAPIEROK
1988/. It was
essential to
ensure tested
insects to be as
homogenous as
possible (the
same
developmental
stage, age, and
physiological
state) in order to
reduce the ________________________________________________________________
sources of Figure 6. Device to test the virulence of P. neoaphidis against the pea aphid
73
variability in a response of specimens to exposure to conidia. Larvae of pea aphid in the

second or the third instar were used in the experiment.
A sterile 50mm-diameter Petri dish bottom lined with a ring of water-soaked sterile filter
paper was used as an inoculation chamber. Disease-free aphids were gently placed into the
dish and directly exposed to the spore shower from inverted sporulating culture (Figure 6). A
fine nylon mesh was set between the bottom and the sporulating culture to prevent the insects
from making a direct contact with culture, but allow the conidia to fall through. During the
shower, the sporulating culture was rotated 90° after each quarter of the exposure time so that
all portions of the inoculation chamber received as nearly equal doses of conidia as possible.
A cover glass was placed in the Petri dish to receive discharged conidia from the plate.
Altogether a group of 20 individuals were used per one dish with a replicate rate of three. For
the test four different exposure times were used at each replicate (e.g. 4 min., 16 min., 60
min., and 180 min.) to obtain a different doses of inocula. The times of exposures could be
adjusted according to the intensity of sporulation of isolates.
Controls (20 aphids) were carried out simultaneously. They were treated similarly, but
aphids were not exposed to a conidial shower.
4.3.5.4 Establishing of the most favourable conditions to infection during and after the
exposure
a. inoculation incubation
Saturated humidity is considered to be generally a basic requirement of the
entomopathogens for sporulation and conidium germination. A moist filter paper placed on
the bottom of Petri dish ensured saturated atmosphere for conidiogenesis inside the
inoculation chamber.
b. post-inoculation incubation
Immediately after the exposure, the sporulating culture and the mesh were removed. The
aphids from the dishes were placed on potted plantlets of pea in plastic boxes (Figure 6). The
insects were maintained for a further time in a saturated atmosphere at 20 ± 2°C and a 16 h
photoperiod. To ensure 100% relative humidity necessary for spore germination, a wet filter
paper was placed on the bottom of the boxes. Controls were treated in the same way.
4.3.5.5 Regularly monitoring of aphid mortality

Daily observations of the insect mortality were made during next consecutive 10 days.
Live and dead aphids were recorded every day and dead ones were removed from the box
before pathogen’s sporulation to avoid cross infection. The cadavers were kept individually in
a small Petri dish with a moist support until the sporulation started. The cadavers were then
examined under microscope to confirm a fungal infection.
4.3.5.6 Establishing the dose-mortality relationship

Measured parameters in the bioassay were a number of aphids exposed to the conidia, a
number of dead infected aphids, mortality (relative ratio of dead treated aphid number and a
number of insects exposed to the conidia), and concentrations of conidia received by aphids.
The conidial concentration was determined in a microscope as a mean number of conidia per
square millimetre based on the counts of conidia from 10 random fields of the cover glass (a
visual field of 0.442 mm2) exposed to the spore shower.
Mortality percentages were probit-transformed and inoculum doses were log-transformed.
If there was any mortality in the controls, treatment data were altered using Abbott’s
correction ((mortality after exposure – mortality in control / 100 – mortality in control) x
74
4. MATERIALS AND METHODS: 4.4 Regular monitoring of aphid populations and Entomophthorales…
100). The subsequent regression line of probit mortality versus log-dose allowed the
estimation of LC 50 (a concentration of conidia killing 50% of the treated insects), which
characterized the virulence of a strain at the time of the experiment /FINNEY 1971/.
A one-way ANOVA (MS Excel 2000) was performed on the LC 50 values to determine if
there were significant differences between isolates. Comparisons of means were performed
using Tukey’s honestly significant different (HSD) test at the 5% level (Statgraphics 4.0).
4.4 REGULAR MONITORING OF APHID POPULATIONS AND ENTOMOPHTHORALES AT DOLNÁ

MALANTA
4.4.1 CHARACTERISTIC OF LOCALITY
Studies to examine aphid population dynamics and fungal mortality of aphids were
conducted on fields and non-cultivated sites at the locality of Dolná Malanta (southwestern
Slovakia, 48°19´ N, 18°09´ E), mostly at the property of Experimental base (EXBA) of
Slovak agricultural university. The Experimental base is situated about 5km east of Nitra.
Nitra belongs to the warmest regions in Slovakia with sum of mean daily temperatures
during main vegetation season (TS10) of > 3000°C and mean daily temperature of 10.2°C
/ŠPÁNIK et al. 2002/. Climatic subregion is characterised as very dry with mild winter. Mean
annual precipitation is 539 mm, and mean annual global radiation is 1336 kWh.m-2 /ŠPÁNIK et
al. 2002/.
Figure 7. Map of sampling sites at the locality of Dolná Malanta
71
The locality consisted of different types of natural and cultivated environments. For
purpose of study four types of habitats were included into sampling of aphids and their fungal
parasites: cultivated plots with annual cultures, non-cultivated sites with trees or shrubs, non-
cultivated sites with ground vegetation forming dense mono-specific patches (e.g. herbs and
weeds), and finally solitary growing herbs or weeds in non-cultivated sites or in cultivated
plots. Anticipating each habitat type might have specific properties to support aphids and their
enemies. Figure 7 provides a map of the study area with sites for aphid sampling displayed.
4.4.2 SAMPLING STRATEGY
The monitoring of aphid populations was carried out during 2001 and 2002 with exception
of stinging nettle patches. The aphids on the nettle and their fungal parasites were sampled
Table 2. Complete list of aphid species and host plants included into sampling at locality D. Malanta
during 2001-2002
Host plant Aphid species Sampling year
Cultivated plots:
Pisum sativum Linné Acyrthosiphon pisum (Harris) 2001, 2002
Triticum aestivum Linné Rhopalosiphum padi (Linné) 2001, 2002
Rhopalosiphum maidis (Fitch) 2002
Metopolophium dirhodum (Walker) 2001, 2002
Sitobion avenae (Fabricius) 2001, 2002
Zea mays Linné Rhopalosiphum padi (Linné) 2001, 2002
Sitobion avenae (Fabricius) 2001, 2002
Faba vulgaris Moench. Aphis fabae Scopoli 2001, 2002
Beta vulgaris Linné Aphis fabae Scopoli 2001, 2002
Hordeum vulgare Linné Diuraphis noxia (Mordvilko) 2001, 2002
Rhopalosiphum padi (Linné) 2002
Rhopalosiphum maidis (Fitch) 2002
Metopolophium dirhodum (Walker) 2002
Sitobion avenae (Fabricius) 2002
Non-cultivated sites:
Malus domestica Borkh. Aphis pomi De Geer 2001, 2002
Dysaphis plantaginea (Passerini) 2001, 2002
Sambucus nigra Linné Aphis sambuci Linné 2001, 2002
Rosa canina Linné Macrosiphum rosae (Linné) 2001, 2002
Chaetosiphon tetrarhodum (Walker) 2001, 2002
Prunus domestica Linné Hyalopterus pruni (Geoffroy) 2001, 2002
Salix sp. Cavariella pastinacae (Linné) 2001
Padus avium Linné Rhopalosiphum padi (Linné) 2001, 2002
Cerasus avium (Linné) Moench Myzus cerasi (Fabricius) 2001, 2002
Acer campestre Linné Periphyllus testudinaceus (Fernie) 2001, 2002
Euonymus europaea Linné Aphis fabae Scopoli 2001, 2002
Cornus sanguinea Linné Anoecia corni (Fabricius) 2001, 2002
Phragmites australis (Cav.) Trin. Hyalopterus pruni (Geoffroy) 2001, 2002
Arctium lappa Linné Aphis fabae subg. mordwilkoi Börner et 2001
Janish
Urtica dioica Linné Microlophium carnosum (Bukton) 2001, 2002
72
during 1998, 1999, 2001, and 2002. At early spring of 2001 the locality was visited and
sampling sites and host plants were chosen to include variety of habitat characters and
diversity of aphids species occupying the habitats. During the study altogether 18 aphid
species were regularly sampled on 19 host plants. Table 2 shows a complete list of aphid
species and host plants, which were included into the regular sampling at given locality.
The monitoring began in mid-March and carried on untill aphid disappeared in the late
autumn. At week intervals the sampling sites were visited and a total of 45 sample units (SU)
were selected at random at each sampling date. Numbers of live aphids and fungus-killed
aphids were counted and recorded by visual inspection of chosen SU. Sample unit in most
cases represented a whole plant. However, in case of cereals a sample unit represented a tiller,
in case of trees or shrubs a terminal 200mm of twig was taken as a SU, a terminal 200mm of
Urtica dioica stems were a SU, and finally for two herbs (Phragmites australis and Arctium
lappa) a leaf was taken as a SU. The sampling was random, however, sampling units were
selected to be as homogenous as possible in size for each species. This was important mainly
for that wild vegetation, where the entire plant or leaf was taken as SU.
When sampling crops, we walked along diagonal transect in each of the plots and
randomly chose sampling plants at approximately 1m intervals on each sampling occasion.
The experimental plots had a size of 1400m2 (35 x 40m) in both years. The crop rotation at
the experimental plots taken in sampling process is displayed in Figure 8.
Aphids that exhibited characteristic symptoms of entomophthoralean infection were
categorised as fungus-killed individuals. Aphids that were living and evinced no visible signs
of disease in situ were categorised as live aphids. Aphids suspected that had succumbed the
infection were carefully removed from the plants and kept inside plastic boxes until infection
verification and pathogen identification. Immediately after collecting, representative samples
of cadavers were microscopically examined. The species of Entomophthorales were identified
and stored as mentioned before (see sections 4.1.1 and 4.1.2). Samples of live aphids were
also collected for species identification (see sections 4.1.3 and 4.1.4). Data for this study was
only collected from the insecticide-untreated plots or sites. χ2-test was used for comparisons
of fungal infections between sampling dates.
In some studies, estimates of fungal infection from aphids colonies in the field were
obtained indirectly by laboratory rearing a certain number of live aphids collected from the
field /e.g. DEAN and WILDING 1973, FENG et al. 1991/. This method seems to reflect more
accurately actual mortality in aphid population at specific time. Since fungus-killed aphids
remain attached to the host plant for certain time, the method of direct counting of cadavers in
the field cannot differentiate between fresh or older aphid cadavers. Moreover, for those
fungal species, which do not produce rhizoids and cadavers are only gently attached to a
plant, certain part of killed aphids can be lost due to their falling of the plant. Despite a lower
accuracy, we used the method of direct counting in situ, since the method of aphid rearing in
the laboratory would have be more labour and time consuming.
Figure 8. Crop rotation at plots included into sampling at EXBA in Dolná Malanta
Plot
I. II. III. IV. V. VI. VII. VIII.
Year
Field Winter Spelt Sugar Spring Field
2001 Maize Alfalfa 2
bean 1 wheat wheat 2 beet barley pea
Field Sugar Spring Field Spring Winter
2002 Alfalfa 2 Maize
pea beet barley bean 1 barley 2 wheat
1
with underseeding of alfalfa; 2 crop was not sampled
73
4.4.3 DATA ANALYSIS FOR SPATIAL PATTERNS AND SAMPLING PLANS

The sample mean ( x , number of fungus-killed aphids and number of live aphids per
sample unit) and variance (s2) (calculated for live and fungus-killed aphids separately) were
calculated for each sampling occasion and host-pathogen-habitat system where epizootic had
developed and these variables were used for other analyses.
Degree of aggregation for live and fungus-killed aphids was determined by the index of
aggregation (λ) that expresses the standard error of mean divided by mean:
(xi − x)
n
∑ 2
λ= =1
(1)
()
i
n (n − 1) x 2
values of index of aggregation (λ) closer to 1 indicate higher aggregation, while values near to
zero indicate more sparse distribution.
The values of mean ( x ) and variance (s2) were related to each other using Taylor’s power
law /TAYLOR 1961, 1971, TAYLOR et al. 1978/ in order to evaluate spatial distribution of both
live and fungus-killed aphids. Taylor’s power law states that the variance of a population is
proportional to a fractional power of the arithmetic mean: s2 = a x b. To estimate a and b, the
values of log 10 (s2) were regressed against those of log 10 ( x ) using model:
log10 (s 2 ) = log10 (a ) + b log10 x () (2 )

where the parameter a is a scaling factor related to the sample size /SOUTHWOOD 1978/, and
the slope b is an index of aggregation, which indicates a uniform, random and aggregated
dispersion when b < 1, b = 1, b > 1, respectively.
The distribution patterns calculated above were confirmed by using χ2-test at 5% level of
significance to test the hypothesis that the mean and variance were equal /ELLIOTT 1997/:
χ 2 = (n − 1) s 2 (3)
2
x
To compare Taylor’s slopes (b) t-test was applied testing the null hypothesis that H 0 : b 1 =
b 2 using a t statistics (4) with df = n 1 + n 2 – 2:
t = (b 1 − b 2 ) (SE 12 + SE )
2 1/ 2
2 (4 )
where b 1 and b 2 are the estimated slopes tested and SE 1 and SE 2 are standard errors of the
slopes.
A functional relationship between the proportion of sample unit bearing fungus-killed

aphids (P 1 ) and the mean density of fungus-killed aphids per SU was developed by assuming
that a negative binomial distribution (NBD) with variable k would describe the distribution of
aphid cadavers on the SU. The NBD-based relationship was chosen because of the close
relationship between NBD and Taylor’s power law /BINNS 1986/. Estimated variance was
74
described as a function of mean /TAYLOR 1961/. With this relationship, k of the NBD can be
calculated:
k= x( ) (a x
2 b
−x ) (5)
The incidence is then one minus the zero term of the NBD /WILSON and ROOM 1983, NYROP
et al. 1989/:
P1 = 1 − 1 [(1 + x k ) ] k
(6)
To generate optimal sample size curves for fungus-killed aphids at fixed levels of
precision, the estimated variance from Taylor’s analysis was incorporated into the usual
expression for standard error of the mean and rearranging:
n = a x b−2 D 2 (7)
where n is the sample size and D is the required level of precision expressed as a proportion
of the mean, and a and b are the coefficients from Taylor’s power law /PENA and DUNCAN
1992, WALKER and ALLSOPP 1993/. We used three values of D; 0.1, 0.3, and 0.5.
4.4.4 TRANSMISSION INFECTION EXPERIMENT

Naturally infected aphids collected from the stinging nettle at Dolná Malanta were used for
transmission experiments to prove a possible horizontal transmission of infection from one
aphid species to another. Microlophium carnosum naturally infected by P. neoaphidis, N.
fresenii, and N. microlophii served as a donor of fungus inoculum in the transmission tests.
Six common aphid species inhabiting crops or non-crop vegetation at the locality,
Acyrthosiphon pisum (Harris), Metopolophium dirhodum (Walker), Rhopalosiphum maidis
(Fitch), Aphis fabae Scopoli, Hyalopterus pruni (Goeffroy), and Macrosiphum rosae (Linné)
were used for the experiment as recipients of the inoculum. The recipient aphids were placed
in quarantine for one week to obtain disease-free individuals. In the course of the quarantine
the aphids were confined in a rearing box at 20 ± 2°C and a 16 h photoperiod. An every-day
monitoring of captured colonies was needful. All dead aphids were removed from the box
before the sporulation could start. Aphids confined in one rearing box originated from the
same colony. After the quarantine period finished all live aphids were considered a disease-
free and suitable for the tests. The recipients were placed inside a small Petri dish (50mm
diameter) and sporulating cadaver of the nettle aphid was adhered on a wet piece of filter
paper on inner side of dish lid. The transmission experiment was carried out in three replicates
for each aphid and fungus species. In each of three replicates, 20 disease-free adult apteral
individuals of each aphid species were exposed to spores shooting off the dead nettle aphid
for about 12 hours. 20 aphids were treated in the same manner, but no cadaver was set on the
filter paper in control variant. After the exposition all the aphids tested were supplied with a
food source and incubated at 20 ± 2°C and a 16 h photoperiod for ten days (see also section
4.3.5.4). During the incubation period the aphids were examined daily. All fungus-killed
aphids were recorded and removed to avoid a cross infection (see also section 4.3.5.5).
The same procedure was carried out to test a susceptibility of six aphid species,
Acyrthosiphon pisum Harris, Diuraphis noxia (Mordvilko), Metopolophium dirhodum
(Walker), Myzus persicae (Sulzer), Rhopalosiphum padi (Linné), and Uroleucon aeneum
(Hille Ris Lambers) to the infection of P. uroleuconii.
75
5. RESULTS AND DISCUSSION: 5.1 Survey of Entomophthorales in aphid populations
5. RESULTS AND DISCUSSION

5.1 SURVEY OF ENTOMOPHTHORALES IN APHID POPULATIONS
A list of 70 aphid species (Table 3) is a result of study on the occurrence of
entomophthoralean pathogens in aphid populations carried out under conditions of Slovakia
and Austria. The aphids listed in the table were found killed by one or more fungi of the order
Entomophthorales in their natural habitats. The list includes aphid species from three different
families. The majority of aphid species belongs to the family Aphididae, altogether 60
species. Out of the family 16 species belong to the tribe Aphidini, 42 species are from the tribe
Macrosiphini, one species is from the tribe Cinarini, and finally one species belongs to the
subfamily Pterocommatinae. Of the family Drepanosiphidae 9 species were susceptible to the
Entomophthorales. Finally, one aphid species susceptible to the pathogens belongs to the
family Anoecidae.
During the survey altogether 15 different entomophthoralean species out of three families
were identified from the aphids in Slovakia. 9 fungal species belong to the family
Entomophthoraceae, 4 species belong to the family Neozygitaceae, and 2 fungi are from the
family Ancylistaceae. Out of the 15 fungal species 9 are the first records from Slovakia, one is
the first record from central Europe, and moreover there is one new species described.
During the study 70 different localities were visited in order to collect the aphid pathogenic
Entomophthorales in Slovakia (Table 1, see section 4.1). A distribution of fungal species
collected at the visited localities in the country is displayed in Figure 9. The pathogens
attacked various aphid populations in all parts of the country involving lowlands, highlands,
Figure 9. Distribution of fungus species recorded from aphids in Slovakia during 1999-2002
76
Table 3. A summary of aphid species and their entomophthoralean pathogens observed in Slovakia and Austria during 1999-2002
Entomophthoraceae: Pandora neoaphidis (Remaudière et Hennebert) Humber

Pandora uroleuconii Barta et Cagáň
Pandora nouryi (Remaudière et Hennebert) Humber
Erynia erinacea (Ben-Ze’ev et Kenneth) Remaudière et Hennebert
Zoophthora radicans (Brefeld) Batko
Zoophthora aphidis (Hoffman in Fresenius) Remaudière et Hennebert
Zoophthora phalloides Batko
Zoophthora occidentalis (Thaxter) Batko
Entomophthora planchoniana Cornu
Ancylistaceae: Conidiobolus obscurus (Hall et Dunn) Remaudière et Keller
Conidiobolus thromboides Drechsler
Neozygitaceae: Neozygites fresenii (Nowakowski) Remaudière et Keller
Neozygites microlophii Keller
Neozygites cinarae Keller
Neozygites turbinata (Kenneth) Remaudière et Keller
Aphidoidea 1. Aphis acetosae subg. acetosae Linné ● ● ●
Aphididae 2. Aphis craccae (Linné) ●
Aphidinae 3. Aphis craccivora Koch ○
Aphidini 4. Aphis fabae Scopoli ● ● ● ●
5. Aphis frangulae gossipii Glover ●
6. Aphis idaei van der Goot ●
7. Aphis nasturtii Kaltenbach ● ○
8. Aphis pomi De Geer ● ● ● ● ●
9. Aphis rumicis Linné ●
10. Aphis sambuci Linné ● ● ●
11. Aphis symphyti Schrank ●
12. Aphis umbrella (Börner) ○ ●
13. Aphis urticata Gmelin ●
14. Hyalopterus pruni (Geoffroy) ● ● ●
15. Rhopalosiphum maidis (Fitch) ○ ● ●
16. Rhopalosiphum padi (Linné) ● ● ● ● ●
Aphidoidea 17. Acyrthosiphon neerladicum Hille Ris Lambers ○
Aphididae 18. Acyrthosiphon pelargonii Kaltenbach ○
Aphidinae 19. Acyrthosiphon pisum (Harris) ● ●
Macrosiphini 20. Brachycaudus cardui (Linné) ○ ● ●
21. Brachycaudus helichrysi (Kaltenbach) ● ●
22. Brachycaudus lychnidis (Linné) ●
23. Brachycorynella asparagi (Mordvilko) ●
24. Brevicoryne brassicae (Linné) ● ● ● ●
25. Capitophorus carduinus (Walker) ●
26. Capitophorus elaeagni (del Guercio) ● ○ ○
27. Capitophorus horni Börner ● ●
28. Cavariella pastinacae (Linné) ● ● ● ●
29. Cavariella theobaldi (Gillette and Bragg) ● ● ● ●
30. Chaetosiphon tetrarhodum (Walker) ●
31. Cryptomyzus gaelopsidis (Kaltenbach) ● ●
32. Cryptomyzus korschelti Börner ●
33. Cryptomyzus ribis (Linné) ● ●
34. Diuraphis noxia (Mordvilko) ● ●
35. Dysaphis plantaginea (Passerini) ●
36. Dysaphis sp. ●
37. Hyadaphis foeniculi (Passerini) ● ●
38. Hyalopteroides humilis Walker ●
39. Hyperomyzus lactucae (Linné) ●
40. Macrosiphoniella artemisiae (de Fonscolombe) ● ●
41. Macrosiphum euphorbiae (Thomas) ●
42. Macrosiphum funestum (Macchiati) ● ●
43. Macrosiphum rosae (Linné) ● ● ● ●
44. Megoura viciae Buckton ● ●
45. Metopolophium dirhodum (Walker) ● ● ● ●
46. Metopolophium festucae (Theobald) ●
47. Microlophium carnosum (Buckton) ● ● ● ● ●
48. Myzus ascalonicus Doncaster ● ●
49. Myzus cerasi (Fabricius) ● ● ● ●
50. Myzus persicae (Sulzer) ● ● ● ● ●
51. Myzus sp. ●
52. Phorodon cannabis Passerini ○
53. Sitobion avenae (Fabricius) ● ●
54. Sitobion fragariae (Walker) ● ●
55. Uroleucon aeneum (Hille Ris Lambers) ● ● ● ●
56. Uroleucon campanulae (Kaltenbach) ● ●
57. Uroleucon erigeronensis (Thomas) ●
58. Uroleucon tanaceti (Linné) ● ● ●
Aphidoidea 59. Pterocomma salicis (Linné) ●
Aphididae
Pterocommatinae
Aphidoidea 60. Cinara pilicornis (Hartig) ● ● ●
Aphididae
Lachninae
Cinarini
Aphidoidea 61. Drepanosiphum platanoidis (Schrank) ●
Drepanosiphidae
Drepanosiphinae
Drepanosiphini
Aphidoidea 62. Callipterinella calliptera (Hartig) ●
Drepanosiphidae 63. Euceraphis betulae Koch ●
Phyllaphidinae 64. Eukalipterus tiliae (Linné) ○ ●
Phyllaphidini 65. Phyllaphis fagi (Linné) ●
66. Therioaphis trifolii (Monell) ●
67. Tuberolachnus salignus (Gmelin) ●
Aphidoidea 68. Chaitophorus leucomelas Koch ●
Drepanosiphidae 69. Periphyllus testudinaceus (Fernie) ● ●
Chaitophorinae
Chaitophorini
Aphidoidea 70. Anoecia corni (Fabricius) ●
Anoecidae
● record from Slovakia and Austria, or from Slovakia only
○ record from Austria only
dryer, and more humid regions. The occurrence of the fungal species varied with regard to
sampling period and sampling area. The fungi could be observed in the aphid populations
from April till November and a great variety of fungal species was found especially during
May and October.
In Austria 7 fungal species were identified from aphid hosts, 4 of them belong to the
family Entomophthoraceae, 2 are from the family Neozygitaceae, and 1 is from the family
Ancylistaceae. The fungi were identified altogether from 32 aphid species. Most of the
samples were collected in Vienna and the federal state of Lower Austria, the regions where
the survey was focused. However, some samples were also collected in the southeast part of
country (Burgenland) or in Upper Austria (the Alp mountains). This study is the first report
on the occurrence of Entomophthorales on aphids in Austria.
5.1.1 DESCRIPTION OF SPECIES COLLECTED DURING THE SURVEY

Pandora neoaphidis:
Pandora neoaphidis was the predominant fungal pathogen of aphids in both the countries.
The species was the most frequently identified in aphid populations. In Slovakia the fungus
was identified, in total, from 48 aphid species that was 68% of the all aphid species. A
complete list of the aphid species, their host plants, localities, and dates of collecting is shown
in the Table 27 (see appendix). The species was encountered from the beginning of April to
the half of November in 60 localities (86%) throughout the country. In some sites an epizootic
level of disease was observed resulting in a collapse of host population. The pathogen infected
a great diversity of aphid species; 72% (35 species) of the aphid species were infesting
herbaceous plants whereas 38% (18 species) of them were infesting trees or shrubs when they
were sampled. Among the group of susceptible aphids important agricultural species also
appeared, including for instance, a cereal-aphid complex (i.e. R. padi, R. maidis, M.
dirhodum, S. avenae, D. noxia), the pea aphid, the black bean aphid, or the green peach aphid.
In Austria the fungus was observed on 26 aphid species (Table 42, see appendix). The
fungus attacked various aphids colonising herbaceous plants (21 aphid species), or trees (7
aphid species). P. neoaphidis was the only pathogen, which was found on aphids from all the
federal states included in sampling and was observed during all sampling periods but one.
Aphids killed by P. neoaphidis were easily distinguished from their living conspecifics in
colonies by an obvious colour change. The cadavers were typically pale brown or brick red,
however, a tinge could vary depending upon the host species (Figure 20a-e). The killed aphids
remained fixed to the plant substrate due to numerous rhizoids, each of which consisted of a
thin stalk ending in a disk-like terminal expansion (Figure 10f-g). Shortly before sporulation
the cadavers became obviously swollen. Primary spores, secondary spores, and some other
fungal structures can be seen in the Figure 10a-f. Biometrics of some taxonomically important
fungal elements is given in the Table 26 (see appendix). No resting spores were observed. The
fungus could be easily isolated and cultivated on culture medium (Figure 21f). Overall, it was
obtained 15 isolates of different origin (Table 25, see appendix).
There is no doubt that P. neoaphidis is truly the most important and most frequent
pathogen observed in aphid populations in Slovakia. It has a worldwide distribution and has
being recorded from nearly all continents; from Europe, Africa, Asia, North and South
America, and Australia /e.g. HALL et al. 1976, MILNER 1982, 1985, FENG et al. 1990, KELLER
1991, STEENBERG and EILENBERG 1995, HATTING et al. 1999a, NIELSEN et al. 2001b, ABDEL-
MALLEK et al. 2003/. In Slovakia the first record of the fungus was probably made in the
course of the pea aphid outbreak on alfalfa in 1921 /VIELWERTH 1921 in STARÝ 1974/, and in
1926 it was recorded again from the aphid in the southern parts of Moravia and Slovakia
/DRASTICH and ROZSYPAL 1927 in STARÝ 1974/. Then there is the work of WEISMAN /1961/
who pointed out a positive role of P. neoaphidis alongside other Entomophthorales species in
77
__________________________________________________________________________________
Figure 10.
Pandora neoaphidis (Remaudière et Hennebert) Humber
a) a cluster of hyphal bodies (aceto-orcein “AO”); a detail of hyphal body (AO); b) primary
conidia with visible nuclei (AO); c) primary conidium with visible nucleus (on the left); secondary
conidia type Ib (on the right) (AO), c, e – the same magnification; d) germinating conidia (AO), b, d –
the same magnification; e) primary conidium forming secondary one, type Ib (on the left); secondary
conidia type Ia (on the right) (AO); f) monohyphal rhizoids with a disc-like endings; g) rhizoids
growing in the ventral part of abdomen of killed aphid.
78
reduction of the black bean aphid population in Slovakia. Recently, studies on the population
dynamics of cereal aphids and the pea aphids as well as their entomopathogens in Slovakia
have been published /ŠTALMACHOVÁ and CAGÁŇ 2000, CAGÁŇ and BARTA 2001/. Results of
those studies indicated a dominant position of P. neoaphidis among other Entomophthorales
identified in the aphid populations. Our work undoubtedly supports the consideration of the
species being the most important aphid pathogenic fungus. We found nearly 70% of aphid
species susceptible to Entomophthorales infected by P. neoaphidis. It is similar to the results
of THOIZON /1970/ who found about 90 aphid species (63%) susceptible to the pathogen in
France. WILDING and BRADY /1984/ stated over 70 aphids on annual and perennial crops,
weeds and wild flowers, from which the fungus was recorded. Comparable results were
obtained during a regional survey of Entomophthorales in Poland /BAŁAZY 1993/ and
Switzerland /KELLER 1991/. The fungus is characteristic of a native propensity for an
epizootic development and a great capacity to a natural control of various aphid populations.
In the course of our survey epizootics were observed for several times, mostly at humid sites
and in thick wild growths. For instance, severe epizootics were observed in populations of M.
carnosum on the stinging nettle, in populations of M. persicae on radish or tobacco crops, in
populations of B. asparagi on an asparagus growth, or in colonies of C. horni on the plume
thistle. The potential for epizootic development in aphid populations has been documented in
number of works /e.g. DEAN and WILDING 1971, MILNER 1985, FENG and NOWIERSKI 1991,
FENG et al. 1992a, NIELSEN et al. 2001a/. As for a fungus seasonal distribution its fully
corresponds with observations of other authors made in the central Europe /e.g. KELLER 1991,
BAŁAZY 1993/. Generally the fungus can be observed in aphid colonies from the beginning of
April to the end of November. The highest number of findings was made during May, June
and October, whereas the least of findings were during August. Correspondingly with other
authors we also did not find resting spores in aphid cadavers. There is only a single
unsubstantiated report of resting spores in P. neoaphidis produced in vitro /UZIEL and
KENNETH 1986/. It was supposed that the fungus may overwinter in the stage of “loriconidia”
/Nielsen et al. 2003/ or specialized hyphal bodies /FENG et al. 1992b/. A plenty of material
collected during the autumn 2002 was microscopically investigated but no conidia or hyphal
bodies like those have been found.
Entomophthora planchoniana:
E. planchoniana was the second most frequent aphid pathogen in Slovakia and Austria. In
Slovakia it was identified from 27 aphid species that was 39% of all the species. The pathogen
was recorded in 14 different localities (20%). All the host aphids, from which the fungus was
identified, localities, and dates of collecting are in the Table 28 (see appendix). Over a half of
the aphid species (15 species, 56%) were feeding on trees or shrubs when the infection was
detected. The pathogen attacked some pestiferous aphids, for example the black bean aphid,
the cereal-aphid complex, the cabbage aphid, the rose aphid, or the green peach aphid. E.
planchoniana was a typical pathogen of Aphis sambuci on elderberry shrubs and the rose
aphid on wild rose shrubs during the spring. It was also frequent on the cereal aphids. A
seasonal dynamics of the disease was the same as that of P. neoaphidis, but no epizootics
were recorded.
In Austria E. planchoniana attacked 10 aphid species mostly on shrubs or trees (7 species).
It was recorded in the majority of visited sites, and during all sampling periods but one. List
of localities and aphid species killed by the pathogen is shown in the Table 43 (see appendix).
Aphids killed by E. planchoniana were light- to dark-brown depending on the colour of
host aphid (Figure 20g) and fixed to the host plant with rhizoids. For morphology of fungus
elements see the Figure 11a-g. Measurements of the fungus structures are shown in the Table
26 (see appendix). We did not observe resting spores despite the structures are known for this
species. Attempts to isolate the fungus failed.
79
__________________________________________________________________________________
Figure 11. Entomophthora planchoniana Cornu
a,b) primary conidia with visible nuclei (aceto-orcein “AO”); c) secondary conidia with a rest of the
primary ones (AO); d) developing conidiophores with visible nuclei (AO); e) conidiophores;
f) branched hyphal body (AO); g) mycelium dissected from a killed cadaver.
80
E. planchoniana is known from Europe, Australia, North and South America, and Africa
/e.g. WILDING 1975, MILNER et al. 1980, FENG and NOWIERSKI 1991a, HATTING et al. 1999a,
2000, ŠTALMACHOVÁ and CAGÁŇ 2000/. It is often encountered in cereal aphid populations
/e.g. DEAN and WILDING 1971, DEDRYVER 1981, 1983, STEENBERG and EILENBERG 1995,
HATTING et al. 1999a, FENG et al. 1991a/ and is frequently reported as a causal agent of
epizootics /e.g. MILNER et al. 1980, COREMANS-PELSENEER et al. 1983, FENG and NOWIERSKI
1991a/. In Slovakia the species was for the first time documented from the black bean aphid
/WEISMANN 1961/ and recently it was listed among pathogens of cereal aphids infecting
maize /ŠTALMACHOVÁ and CAGÁŇ 2000/. According to our results it was frequent on cereal
aphids, the green peach aphid, as well as the rose aphid. It is of interest that despite it was
found on one fifth of all the localities visited, no epizootics were recorded in host populations.
The fungus could be observed individually or together with other entomophthoralean species
as a component of parasitic mycoflora in aphid colonies. If the latter event came about, the
species never reached a dominant position. The majority of findings was made during June
and July and from October to beginning of November. In Yugoslavia SIVČEV /1992/ observed
the fungus in the cabbage aphid populations to be active predominantly during the autumn. In
Slovakia the largest number of aphids was infected during relatively dryer periods. KELLER
/1987a/ also noticed that the fungus preferred relatively dry habitats and did not occur in
dense humid crops. No resting spores were observed inside killed aphids. A reason for this is
unknown. Probably the resting spores are formed just in a certain low percentage of cadavers
and the low proportion of cadavers investigated during the survey was not enough to discover
the spores. Resting spores were not observed by others as well. For instance, the spores were
not find in the South Africa during a survey of cereal aphid pathogens /HATTING et al. 1999a/.
The species is known to produce resting spores /KELLER 2002/ and it is unlikely that the
resting spore formation would have not been induced because of lack of external stimuli.
Under condition of Yugoslavia the resting spores were observed mostly in October and
November /SIVČEV 1992/. We found neither any thick-walled hyphal bodies inside of
cadavers in the autumn or structures similar to those described by KELLER /1987b/ as
alternative overwintering elements of the pathogen.
Neozygites fresenii:
In the course of our survey N. fresenii was identified from 24 aphid species (35%). List of
the aphid species, their host plants, localities, and dates of collecting is shown in the Table 29
(see appendix). The species was recorded from 21 localities (30%) in the country. A seasonal
dynamics of the disease was similar to those of P. neoaphidis and E. planchoniana, but the
infection usually appeared one month later. N. fresenii had a strong tendency to establish
epizootics in dense aphid colonies, especially in those of Aphis fabae, Aphis nasturtii, or
Microlophium carnosum. In addition, the fungus attacked another pest aphids like
Rhopalosiphum padi, Myzus persicae, or Brevicoryne brassicae.
In Austria the fungus infected 10 aphid species. And it was recorded in 8 localities (Table
47, see appendix).
Aphids killed by N. fresenii were brown-black or grey (Figure 20m-o) and they did not
change the shape. When resting spores were formed inside of the host body, the colour of
aphids changed to black at their death (Figure 20l-m). Rhizoids were absent and cadavers
were attached to the plant surface by their proboscis. Cadavers filled with resting spores
liquefied after a period of time and the aphid’s content with resting spores effused onto the
plant surface or dropped to the soil. Specific behaviour modifications were detected for aphids
infected by N. fresenii. We noticed that aphids killed by the fungus left attached to the leaf
blade, i.e. at the feeding site of the aphids, when conidia were formed. But when resting
spores were formed the cadavers were attached to the leaf petioles (for instance A. fabae on
sugar beet, Figure 20m-o), or the plant stem and the leaf petioles (for instance M. carnosum
81
__________________________________________________________________________________
Figure 12. Neozygites fresenii (Nowakowski) Remaudière et Keller
a) capilliconidia (aceto-orcein “AO”); b) detached capilliconidia (AO); c) capillary conidiophores

and detached capilliconidia (AO); d) primary conidium forming three capillary conidiophores and a
rest of primary conidium with a capilla (AO); e,f) premature resting spores (AO); g,h) mature resting
spores with black episporium.
82
on the stinging nettle), although the usual feeding site for the aphid is the leaf blade. Figure
12a-h shows the important taxonomic structures of the species and the Table 26 (see
appendix) includes measurements of fungus structures used for the species identification. No
attempt was made to isolate the fungus, as it has never been successfully cultured in vitro
/KELLER 1997/.
The species is known from all continents including the South Pacific region /KELLER
1997/. It is often considered to be best adapted to tropical conditions /STEINKRAUS et al. 1991,
KELLER 1997/ although it is effective in the subpolar region, as well /AUSTARÅ 1997,
NIELSEN et al. 2001b/. In Slovakia the species infected more than one third of the aphid
species discovered to be susceptible to Entomophthorales. The fungus infected principally
aphids living in dense colonies and epizootics were often observed in those aphid colonies
what underlines a significance of the pathogen as a natural control agent. After the first
infected aphids appeared within colonies, the disease usually developed into strong epizootics
very fast and within few days entire colonies were destroyed. If a coexistence of N. fresenii
with more entomophthoralean species was present in colonies, N. fresenii usually gained
dominance over the other fungus species in a short time. The great effectiveness of the species
was documented by many authors /e.g. DEDRYVER 1978, STEINKRAUS 1991, SMITH and
HARDEE 1996, NIELSEN et al. 2001b/. The species was identified from less number of host
species when compared with E. planchoniana, but it was recorded in more localities. This
may imply narrower host spectrum but higher activity of N. fresenii. However, this may also
relate with different types of niches occupied by both species. While N. fresenii was frequent
in humid habitats at ground dense vegetation, E. planchoniana infected aphids living in
relatively dryer microclimates at upper levels of vegetation. Aphid cadavers killed by N.
fresenii were often found being colonised by saprophytic mycoflora what is in accordance
with observations of other authors /e.g. STEINKRAUS et al. 1991/. RONG and GROBBELAAR
/1998/ reported that some cadavers of Aphis gossypii harboured both N. fresenii and a species
of Cladosporium sp. ROBERTS and HUMBER /1981/ and HUMBER /1991/ noted that species of
genera Cladosporium, Fusarium, and Penicillium are often encountered as necrophytic
saprobes on aphid cadavers and are, at best, very weak pathogens of aphids. Cadavers with
resting spores were generally observed during dry and hot periods in the summer and were
less common during the autumn. However, SIVČEV /1992/ found the spores in dead aphids
only in the autumn. In the literature there are no reports about a change of behaviour of aphids
infected with N. fresenii. There is only a note of SIVČEV /1992/ who found that the cabbage
aphids infected with this fungus were usually found on the leaf petioles or at the beginning of
the main leave vein. But he did not mention if the aphids were filled with resting spores or
were producing conidia. We found the behavioural modification only for aphids with resting
spores. We assume that this phenomenon is a great example of a fungus adaptation to survive.
It presumably facilitates or increases the probability of the resting spores to get to the soil.
Conidiobolus obscurus:
C. obscurus is next important aphid pathogenic fungus. In Slovakia it infected 17 aphid
species (25%). All the aphids belonged to single family, the family Aphididae (Table 30, see
appendix). The fungus was recorded in 13 localities, that was 19% of all the localities visited
in the country. It was mainly observed in aphid colonies feeding on herbaceous plants (82%).
Like the previous fungi, C. obscurus also contributed to a reduction of abundance of different
pestiferous aphid species, for instance of the black bean aphid, the cereal aphids, or the pea
aphid. The infection was normally recorded during June and October. The fungus was present
within aphid colonies at low levels alongside other fungi as an occasional species and the
disease development never reached epizootic character. At the end of vegetation period
resting spores were usually produced.
83
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Figure 13. Conidiobolus obscurus (Hall et Dunn) Remaudière et Keller
a) primary conidia (aceto-orcein “AO”); b,c) conidiophores with forming primary conidia (AO); d)
germinating conidia forming germ tubes or secondary conidia (AO); e) hyphal bodies (AO); f) mature
resting spores with thick cell wall(AO); g) premature resting spores (AO).
84
In Austria the fungus was identified from three aphid species, A. pisum, A. umbrella, and
R. padi (Table 44, see appendix) with low infection levels.
Aphids killed by C. obscurus were grey to greyish brown in colour, and attached to plants
by proboscis head downwards (Figure 20i-j). At sporulation the colour of cadavers turned into
white. For morphology of the fungus see the Figure 13a-g and biometrics is presented in the
Table 26 (see appendix). The fungus can be easily isolated and cultivated on artificial media
(Figure 21i). One isolate was obtained from the green peach aphid (Table 25, see appendix).
Despite the species is quite frequently encountered within aphid populations in Slovakia,
its ability to cause epizootics in host populations seems to be low. This is in agreement with
KELLER and SUTER /1980/ who say that though C. obscurus is common in Switzerland its
tendency to cause epizootics is not as prominent as for P. neoaphidis. The fungus was only
sporadically observed in aphid colonies as a single disease cause. It usually acted along with
P. neoaphidis, E. planchoniana, or N. fresenii. This is also known from many examples in the
literature /e.g. DEAN and WILDING 1971, WILDING 1975, COREMANS-PELSENEER 1981,
DEDRYVER 1981, SIVČEV 1992, CAGÁŇ and BARTA 2001/ and infection usually do not
surpassed a level of 30% in populations /e.g. WILDING 1975, DEDRYVER 1981, FENG and
NOWIERSKI 1991a, PICKERING and GUTIEREZ 1991, SIVČEV 1992, CAGÁŇ and BARTA 2001/.
In Slovakia it was observed for the first time in colonies of the black bean aphid /WEISMANN
1961/ and later in the pea aphid colonies /CAGÁŇ and BARTA 2001/. From the viewpoint of
aphid control C. obscurus is considered not as important natural aphid control agent as the
previous species under conditions of Slovakia.
C. obscurus closes the foursome of the most important aphid pathogenic Entomophthorales
characterised by a worldwide distribution, an obligate specificity to aphids, a pathogenicity to
rather great variety of aphid species, a high prevalence in aphid colonies, and finally by an
ability to cause epizootics in host populations. Following species are usually considered a
minor pathogen of aphids for various reasons, e.g. for a low specificity to aphids or, on the
other hand, an obligate specificity to single aphid species, an absence of epizootics, or for a
rare presence in aphid populations.
Conidiobolus thromboides:
C. thromboides was identified from one aphid species, the spotted alfalfa aphid (Table 37,
see appendix) and was not observed in Austria. The fungus was recorded in two localities in
the south part of the country at the end of summer. This report is the first record of C.
thromboides from Slovakia.
Aphids killed by C. thromboides were brick red coloured and attached to leaflets of alfalfa
with monohyphal rhizoids (Figure 14b). Most of the cadavers were situated in the lower third
of the alfalfa plants and the aphid bodies were attached to the underside of leaflets. Some
fungal structures, primary and secondary conidia or conidiophores, can be seen in the Figure
14a-e. Dimensions of conidia are shown in the Table 26 (see appendix). Aphids filled with
resting spores were not observed. The fungus could be isolated in vitro (Figure 21g) and 3
isolates were successfully obtained on SDAYEM (Table 25, see appendix).
Despite the absence or presence of rhizoids for this taxon was discussed by HUMBER et al.
/1977/ and it was concluded that the species did not form the structures we did find the
cadavers firmly attached to substrate with hyphae resembling to rhizoids. They were
monohyphal, thick, free of cytoplasm, and with enlarged endings. REMAUDIÈRE et al. /1976b/
suggested that the rhizoids noted originally by HALL and DUNN /1957/ might have been
observed from aphids which were infected simultaneously by another fungus, which did
produce rhizoids and did not produce conidia. This theory was fully accepted later by
HUMBER et al. /1977/. It seems unlikely that the same case would have been recorded in
Slovakia, however it is in all probability not impossible. Unfortunately we could not study the
85
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Figure 14. Conidiobolus thromboides Drechsler
a) primary conidia and germinating primary conidia(aceto-orcein “AO”); b) monohyphal rhizoid c)

secondary conidia with a rest of the primary one (AO); d) primary conidium forming atop of simple
conidiophore; f) germinating conidia forming successive conidia (AO).
86
discrepancy more precisely because of lack of material and even the isolates sporulated poor
so that aphids could not be infected experimentally.
Prevalence of infection in the aphid population was low and only few infected cadavers
were collected in the colonies of the spotted alfalfa aphid in Slovakia. Even though the
species is reported all over the world mostly as a pathogen of aphids /BAŁAZY 1993/ and was
intensively studied for possibilities of aphid control /ČUDARE 1990/ its value as a natural
control agent was low in Slovakia. Epizootics were not observed in natural habitats, however
experimental applications with resting spores usually resulted with high infections in tested
populations /KREJZOVÁ 1972a, SOPER 1978, ČUDARE 1990/. It is not clear why the species
was of such a low effectiveness in Slovakia. The fungus is mostly identified from cereal aphid
/FENG et al. 1990, FENG and NOWIERSKI 1991a, STEENBERG and EILENBERG 1995, HATTING
et al. 1999a, ABDEL-MALLEK et al. 2003/. The fungus was for the first time described from
Therioaphis maculata on alfalfa in California /HALL and DUNN 1957/, i.e. the same aphid
from which the fungus was identified in Slovakia.
Pandora nouryi:
P. nouryi was identified from one aphid species, Aphis acetosae, in Slovakia (Table 38, see
appendix), and was not observed in Austria. The species was recorded in one locality in
October. This is the first report of P. nouryi from Slovakia.
There was only single colony of the host aphid found to be infected by the fungus. The
aphid dwells inside of curled leaves of Rumex sp. and the cadavers were firmly attached to the
underside of leaf with rhizoids. The infected colony was situated at the ground level, whereas
colonies at the upper part of plant were not infected. Within the colony 14 killed aphids were
recorded and among them over a half were filled with spherical resting spores. Morphological
features of the fungus are shown in the Figure 15a-f. Because of lack of sporulating cadavers
no attempt was made to isolate the species.
The A. acetosae is a new host for the species. P. nouryi is known only from Europe and it
mostly infects root dwelling aphids especially those from the genus Pemphigus. BAŁAZY
/1993/ says that the occurrence of Pandora nouryi on root aphids Pemphigus bursarius and P.
phenax has been observed in the North-Western and Central Europe as well as in Israel.
MAJCHROWICZ /1999/ made a survey on the occurrence of Entomophthorales on P. bursarius,
P. phenax, Dysaphis crataegi in Poland and P. nouryi was predominant pathogen of the
aphids. On the other hand the species can also be identified from leaf aphids from the genus
Aphis /GUSTAFSSON 1965, MILNER 1981/.
Erynia erinacea:
Aphids killed by E. erinacea were observed in Slovakia and Austria from Aphis umbrella
feeding on Malva sp. (Tables 39 and 48, see appendix). The species was recorded from only
one locality in both countries. The fungus attacked the host colonies at the end of summer and
fungal prevalence had in the both localities an enzootic character. This is the first record of E.
erinacea from the central Europe.
Aphids killed by the fungus were light brown in colour and the cadavers were attached to
substrate with their proboscis. Rhizoids were not observed. In Austria only cadavers filled
with echinulate resting spores were collected (Figure 16a-b). However, in Slovakia some
specimens with sporulating fungus were found within a single colony. See the Table 26 for
dimensions of primary conidia and resting spores. Because of lack of material no attempt to
isolate the species was made.
Until now the species has been known from Israel where the fungus attacked aphids from
the genus Aphis and M. persicae /BEN-ZE’EV and KENNETH 1979/. The fungus was described
in Israel in 1978 and was not reported during the following 25 years anywhere else.
87
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Figure 15. Pandora nouryi (Remaudière et Hennebert) Humber
a) primary conidia (aceto-orcein “AO”); b) hyphal bodies (AO); c,d,g) formation of resting spores
(AO); e) premature resting spores (AO); f) mature resting spores (AO).
88
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Figure 16.
a,b) mature resting spores with echinulate walls (aceto-orcein “AO”).

c) hyphal bodies d) conidiophores; e) primary conidia.
89
Zoophthora aphidis:
Z. aphidis was identified from 4 aphid species in Slovakia that was 6% of all the aphid
species. All the cadavers were found in one locality (Table 33, see appendix). Among the
aphid species 3 important pest aphids were recorded, R. padi, A. pomi, and M. cerasi. The
infection was rare within these aphid populations and its prevalence did not exceed a level of
1%. The infection was typical of its type host, sexuales of A. corni, feeding on leaves of red
dogwood. The infection appeared mostly during October in populations of the type host or the
other above mentioned aphid species, and has never been observed during spring or summer.
This report is the first record of Z. aphidis from Slovakia.
In Austria the fungus was observed in populations of A. corni in three localities (Table 45,
see appendix).
Aphids killed by Z. aphidis were creamy white and sporulation was restricted to dorsal
region in one or few delimited areas. Mostly the alate individuals were killed and white
clusters of sporulating conidiophores usually appeared in intersegmental zones or between
thorax and abdomen (Figure 20h). Cadavers were firmly attached to the plant with rhizoids.
Figure 17a-d shows morphology of the fungal structures. For biometrics of the fungal
structures see Table 26 (see appendix). Resting spores were not observed. We did not try to
isolate the fungus onto artificial media.
The fungus is reported from Europe mostly from its type host A. corni /MILNER 1981,
KELLER 1987a, BAŁAZY 1993/. BAŁAZY /1993/ stated that attempts to find this fungus in
populations of Anoecia sp. on Cornus sanguinea L. in Poland, Romania, Eastern France or
Germany had been unsuccessful for last 2 decades. Our results are in accordance with
observations of other authors. The fungus appears at the end of September and during October
in populations of alate sexuales /KELLER 1987a/. KELLER /1987a/ observed cadavers with
sporulating fungus fixed by rhizoids to the underside of leaves, whereas those individuals
filled with resting spores were mainly around the buds in the leaf shoulders or also along the
main veins of the leaves. We did not observed resting spores in the cadavers, however the
cadavers with sporulating fungus could be found on either abaxial or adaxial side of leaves
mostly along the main veins. Majority of killed individuals were alate ones, only few apterous
aphids were killed. The fungus was not common in Slovakia, although it appeared on A. corni
at the locality each year on the same shrub. A. pomi and M. cerasi are new hosts for the
pathogen, whereas R. padi was found to be infected by the species in Switzerland as well
/KELLER 1987a/. The cadavers of R. padi, A. pomi, and M. cerasi were found once at the same
collecting site where the infected colonies of A. corni were present.
Zoophthora radicans:
Z. radicans was observed on three aphid species, B. brassicae, Ch. leucomelas, and M.
persicae in three localities in Slovakia (Table 32, see appendix). It was not recorded in
Austria. The pathogen was not frequent in aphid populations, however, there was an epizootic
observed within colonies of the cabbage aphid on winter rape in 2002. Simultaneously a
population of Plutella xylostella Linné infesting the same field was severely infected with Z.
radicans and Pandora blunckii (Lakon ex Zimmermann) Humber. It was not studied whether
both the pest species were infected by the same strain of Z. radicans or strains with different
specificity appeared at that site. This report is the first record of Z. radicans from Slovakia.
Aphids killed by Z. radicans were light brown and attached to the plant surface with
rhizoids. For morphology of primary and secondary conidia see the Figure 17e-i. Table 26
(see appendix) shows results of measuring of the fungal structures. Cadavers filled with
resting spores were not observed. The species can grow on artificial media (Figure 21h) and
one isolate was obtained (Table 25, see appendix).
Z. radicans is one of the entomophthoralean fungi, which is characterised of extraordinary
wide host range /KELLER 1991, BAŁAZY 1993/. The species can be sporadically observed on
90
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Figure 17.
Zoophthora aphidis (Hoffman in Fresenius) Remaudière et Hennebert
a,b) primary conidia with visible nuclei (aceto-orcein “AO”); c) primary conidia forming
capilliconidia and a rest of primary conidium with a capilla (AO); d) detached capilliconidia (AO).

e,f) primary conidia with visible nuclei (AO); g) primary conidium forming capilliconidium (AO);
h) detached capilliconidia (AO); i) detached capilliconidia and rests of primary conidia with capillae
(AO).
91
aphid hosts and a level of infection is usually low /e.g. FENG et al. 1990, KELLER 1991,
SIVČEV 1992/. The pathogen attacks aphids from different families, for instance Aphididae
/FENG et al. 1990, SIVČEV 1992/, or Drepanosiphidae /KELLER 1991, MILNER and SOPER
1981, MILNER et al. 1982/. During our survey the fungus was present in the host populations
in a very low prevalence, however, in the cabbage aphid colonies the infection developed into
epizootic. Resting spores are rarely formed in vivo /FENG et al. 1990, SIVČEV 1992/.
Zoophthora phalloides:
Z. phalloides was identified from C. pilicornis on spruce trees in Slovakia (Table 40, see
appendix). Within dense colonies of aphid the fungus killed a few alate individuals of the host
aphid at the beginning of July and was never observed later. The species was not found during
a survey of aphid populations in Austria. In Slovakia the fungus was found for the first time.
Aphids killed by the fungus were brown in colour and attached to needles by rhizoids.
There are shown hyphal bodies, conidiophores and conidia in the Figure 16c-e. For biometrics
of the fungal structures see Table 26 (see appendix). Resting spores were not observed. We
did not try to isolate the fungus onto artificial media.
Z. phalloides is an obligate aphid pathogenic fungus with a worldwide distribution, but the
fungus is not frequently encountered in aphid colonies /BATKO 1966a, REMAUDIÈRE et al.
1976, 1981, BEN-ZE’EV et al. 1984, GLARE et al. 1985, KELLER 1991, NIELSEN et al. 2001a/.
In Slovakia few cadavers were observed in colonies of C. pilicornis in one locality. The aphid
is a new host for the fungus. It was observed that the species prefers aphid colonies in natural
vegetation /KELLER 1991, NIELSEN et al. 2001a/ and is less often in annual crops
/REMAUDIÈRE et al. 1981/.
Zoophthora occidentalis:
Z. occidentalis was identified only from one aphid host, U. tanaceti, in one locality in
Slovakia (Table 41, see appendix). This is the first record of Z. occidentalis from Slovakia.
The fungus was not found in Austria.
External symptoms of Z. occidentalis infection were similar to those of Z. radicans. For
dimensions of primary conidia see Table 26 (see appendix). Resting spores were not observed
and no effort was made to isolate the fungus because of lack of material available.
Z. occidentalis is another obligate aphid pathogenic species spread out in North and South
America, Europe and Asia on several, mostly on unidentified aphid species /BAŁAZY 1993/.
The fungus is rare and is not known to create epizootics. In Europe the pathogen was
identified from aphids on the stinging nettle /BATKO 1962/, Anoecia sp. /THOIZON 1970/,
aphid on Deschampsia ceaspitosa (Linné) /MIETKIEWSKI et al. 1981/, Capitophorus similis
van der Goot, Impatientium asiaticum (Nevsky) and Sitobion fragariae (Walker) /BAŁAZY
1993/. U. tanaceti is a new host for the pathogen.
Neozygites microlophii:
N. microlophii was identified from its type host, M. carnosum, on the stinging nettle. The
species was found in one locality in Slovakia (Table 35, see appendix) and not in Austria. The
infection occurred during May and June and there was an epizootic developed within the host
populations in 1999. Each year the species appeared together with N. fresenii in the aphid
colonies and both species significantly influenced a development of host populations. The
species is reported from Slovakia for the first time.
External symptoms of N. microlophii infection were similar to those of N. fresenii. Dead
aphids were fixed to plants by proboscis (Figure 20k). No rhizoids were formed. Cadavers
were gray when fungus sporulated or black when resting spores were formed in the aphid
bodies. Cadavers filled with resting spores liquefied like the aphids killed by N. fresenii.
Similarly the changes in aphid behaviour like in the case of N. fresenii were also noticed.
When resting spores were formed the aphid moved to a plant stem shortly before its death.
92
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Figure 18.
a) primary conidia; b); conidiophores with forming primary conidia; c) a rest of primary conidium
with a capilla; d) detached capilliconidia; e) mature resting spores.

f) mature resting spores with a germ capilliconidium (arrow); g) resting spore with a germ capilla;
h) resting spore with a germ capilliconidium; i) detached germ capilliconidia, arrow – a drop of sticky
fluid.
93
Figure 18a-e shows the important taxonomic structures of the species and the Table 26 (see
appendix) summarizes measurements of the fungus structures used for the species
identification. No attempt was made to isolate the fungus.
The fungus is a monophagous species pathogenic to M. carnosum, the aphid creating dense
colonies on the stinging nettle /KELLER 1991/. The species was described in Switzerland
/Keller 1991/ and resting spores corresponding with those of this species were observed inside
of cadavers of the nettle aphid in Poland as well /BAŁAZY 1993/. The species has not been
probably reported from other countries. As was noticed by KELLER /1991/ the fungus could
create a strong epizootics in the host populations. In Slovakia the species was discovered in
one locality, but there is no doubt that the fungus is common in the nettle aphid populations.
Neozygites cinarae:
Some individuals killed by N. cinarae were observed in colonies of its type host aphid, C.
pilicornis, on spruce twigs. The pathogen was recorded in two localities in Slovakia and one
locality in Austria during July or August (Tables 36 and 46, see appendix). No epizootics
were observed at the localities. This fungus is reported from Slovakia and Austria for the first
time.
Aphids killed by N. cinarae were grey to light brown and attached to the twigs by
proboscis. No rhizoids were observed. Some individuals were black in colour. These were
filled with black oval resting spores. For morphology of taxonomically important fungus
structures see the Figure 19a-f. Biometrics of the structures is in the table 26 (see appendix).
No attempt was made to isolate the fungus.
Until known the fungus has been known only from Switzerland /KELLER 1997/. The
fungus seems to be common in the aphid populations, but epizootics were not observed.
Neozygites turbinata:
A strong epizootic was observed in colonies of the giant willow aphid feeding on branches
of willow trees during October 2001. The outbreak resulted in nearly extinction of the host
population. The infected aphids were filled with black ovoid resting spores typical of the
genus Neozygites. A cause of the disease was attributed to a pathogenic fungus identified as
N. turbinata. Cadavers filled with resting spores left hanging on their proboscis from the
branches for several weeks (Figure 20a-b). After certain time the cadavers either dried up and
fell down to the soil or they liquefied and remainders of resting spore mass left on a bark of
the trees. Figure 21c-e shows the remainders of resting spores three months after the epizootic
on the willow twigs. Some detectable residues of the resting spores were found on the trees
even after one year. No cadavers with sporulating fungus were found. N. turbinata was
identified from two aphid species in two localities in Slovakia (Table 34, see appendix). This
is the first report of the fungus from Slovakia and it was not found in Austria. Figure 18f-i
shows the resting spores and germ conidia of the species. Measurements of the structures are
shown in the Table 26 (see appendix). We did not try to isolate the fungus on artificial media.
The species was originally described from Pterochloroides persicae (Cholodk.) in Israel
/KENNETH 1977/. 20 years later the fungus was rediscovered in Switzerland on aphid
Tuberolachnus salignus Gmelin on Salix sp. where the fungus caused strong epizootic during
September and October /KELLER 1991/. In Slovakia the fungus was first identified from aphid
Pterocomma salicix (Linné) on Salix sp. and later the epizootic in the colonies of T. salignus
was recorded. The epizootic outbreak was noted in October and cadavers continued to hang
on branches through November until the winter. The epizootic was observed only in one
locality although in the following year many willow trees were watched purposely to find the
infection, but neither aphids nor infection were observed. Pterocomma salicix is a new host
for the fungus.
94
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Figure 19. Neozygites cinarae Keller
a) primary conidia; b) conidiophores; c) primary conidium germinating and forming a germ tube; d)
primary conidia; e) primary conidium forming a capilliconidium and rests of primary conidia with
capillary conidiophores; f) resting spores dissected from a host.
95
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Figure 20.
a) the pea aphid killed by P. neoaphidis, white circle of projected conidia around the cadaver b) the
common nettle aphid killed by P. neoaphidis; c) Uroleucon aeneum killed by P. neoaphidis; d,e) aphids
killed by P. neoaphidis are fixed to plant by rhizoids forming on the ventral part of abdomen (arrows);
f) a group of Uroleucon aeneum killed by P. uroleuconii; g) the rose aphid killed by E. planchoniana;
h) Anoecia corni killed by Z. aphidis; i,j) the common nettle aphid killed by C. obscurus; k) the
common nettle aphid killed by N. microlophii; l,m) cadavers of the black bean aphid filled with resting
spores of N. fresenii on petioles of sugarbeet; n) a colony of the black bean aphid infected by N.
fresenii; o) the common nettle aphid killed by N. fresenii.
96
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Figure 21.
a, b) cadavers of the giant willow aphid filled with resting spores of N. turbinata and hanging down
a willow branch; c,d,e) rests of the aphid cadavers with black resting spores on willow branches after
six months.
Plates of medium with growing cultures of P. neoaphidis (f), C. thromboides (g), Z. radicans (h),
and C. obscurus (i).
97
We found a great species diversity of entomophthoralean fungi attacking aphids in

Slovakia and Austria. The 15 entomopathogenic fungi observed during the survey surpass the
number of pathogens reported from the aphid fauna by other authors in different countries or
regions all over the world. Regional list of aphid pathogenic fungi have been published for
instance in Sweden /GUSTAFSSON 1965/, France /THOIZON 1970/, Australia /MILNER et al.
1980/, Israel /BEN-ZE’EV et al. 1984/, Finland /PAPIEROK 1989/, Poland /BAŁAZY et al. 1990/,
South Africa /HATTING et al. 1999a/, or Iceland /NIELSEN et al. 2001b/. Entomophthoralean
fungi were reported from 34 aphid hosts in eastern Canada and the USA /REMAUDIÈRE et al.
1978/. HALL and DUNN /1957/ mentioned that 10 fungal species were found on aphids in
California and included 4 newly described species, however some of those are at present
considered synonymous. THOIZON /1970/ listed from France 142 aphid species susceptible to
15 entomophthoralean fungi, however 3 of them are at present considered synonymous and
one was later rejected. Seven entomopathogenic fungi from the order Entomophthorales were
recorded on 15 aphid species during a survey of aphid pathogens in restricted region in Poland
/BAŁAZY et al. 1990/.
Up to now 31 entomophthoralean fungus species have been known to infect aphids in
various parts of the world. Some of them have a cosmopolitan distribution, however, there are
species that have their geographical distribution restricted to their type locality, or some are
poorly or incompletely described in this respect. Out of the group of 31 species 23 fungi have
been recorded from Europe. Aphid-fungal pathogen associations in Slovakia have not been
documented well with only few reports published. Up to now 5 entomophthoralean species
have been observed on aphids in this country. They were P. neoaphidis, E. planchoniana, N.
fresenii, C. obscurus, C. destruens /WEISMANN 1961, STARÝ 1974, ŠTALMACHOVÁ and
CAGÁŇ 2000, CAGÁŇ and BARTA 2001/. However, C. destruens did not infected aphids in
nature, it was originally described from cadavers of gnats during the seventies of the last
century /WEISER and BATKO 1966/ and it showed pathogenicity to aphids in laboratory
bioassays /KREJZOVÁ 1972a/.
All the seven entomophthoralean species found in Austria are considered the first record
from the country. 10 of the group of 15 species from Slovakia are the first record in the
country. Most of the entomophthoralean fungi listed in the Table 3 are distributed worldwide
so there is no surprise that they were identified from aphids in Slovakia and Austria. We
found a set of four entomophthoralean species to be the most important pathogens in both the
countries. They were P. neoaphidis, E. planchoniana, N. fresenii, and C. obscurus. These
species were the most frequent in aphid colonies and they were identified from majority of
aphid species. Plenty of findings of entomophthoralean fungi were from agricultural crops
including the most important pestiferous aphids as hosts for the pathogens, e.g. A. pisum, M.
persicae, R. padi, R. maidis, M. dirhodum, S. avenae, D. noxia, and A. fabae. This was
because fields were specifically targeted in our survey.
Surprisingly, some species, which have been up to now recorded from one or few localities
in the world were observed during the survey, as well. They were N. microlophii, N. cinarae,
N. turbinata, and E. erinacea. On the other hand there are species, which are usually found in
aphid colonies, but they were not observed in Slovakia or Austria. C. coronatus is a typical
example. Despite C. coronatus is considered to live mainly as a widespread soil saprophyte,
the species is known to cause disease not only in insects of different orders but also in
mammals /MACLEOD and MÜLLER-KÖGLER 1973, KELLER 1987a/. The species is often
isolated from soil samples /e.g. COREMANS-PELSENEER et al. 1983, SAJAP et al. 1997, ALI-
SHTAYEH et al. 2002/ and can attack insects inhabiting the soil /KELLER 1987a/. Many
investigations suggest that C. coronatus does not play a significant role in regulation of aphid
populations /e.g. REMAUDIÈRE et al. 1981, PAPIEROK 1985, HATTING et al. 1999a/. Under
natural conditions it can be regarded as a non-primary pathogen invading secondarily insects,
98
which have died from another cause /PAPIEROK 1986/. Although, it has been found to attack
leaf aphids, as well /e.g. BATKO 1962, THOIZON 1970, PAPIEROK 1985, SIVČEV 1991,
HATTING et al. 1999a/. Another mostly saprophytic Conidiobolus species, which was not
recorded during our survey, is C. osmodes. This species was able to manifest itself in aphid
populations at epizootic levels in France /PAPIEROK and COREMANS-PELSENEER 1980/.
Remaining aphid pathogens, which were not observed during our study, are either species
with distribution outside of Europe, e.g. E. chromaphidis, P. delphacis, P. kondoiensis, N.
lecanii, or Z. anhuiensis, or are only rarely identified from aphids, e.g. B. apiculatus, B.
major, E. atrosperma, or N. lageniformis /BAŁAZY 1993, KELLER 1997/. THOIZON /1970/
mentioned E. conica on aphids from France. Besides this isolated record this species is known
only from Diptera: Nematocera, mainly belonging to the families Chironomidae and
Culicidae /e.g. Keller 1991, Bałazy 1993/. Since the Entomophthorales are known for their
host specificity, the record of E. conica on aphidis must be considered doubtful and it needs
confirmation.
It was noted that among the entomophthoralean species recorded during the study the
resting spores were rarely produced or were not observed at all. Only Neozygites species
regularly produced resting spores inside the cadavers mostly in August or September. C.
obscurus was another species producing resting spores commonly in the autumn. It is not
clear why no resting spores were observed in other species. Perhaps we did not collect
adequately to observe any or more cadavers with resting spores. In Israel /BEN-ZE’EV et al.
1984/ and the USA /FENG et al. 1990/ the resting spores of Entomophthorales were also quite
rare during a survey of arthropod-pathogenic fungi.
The four-year study results conducted in Slovakia and Austria clearly indicate that the
group of fungi regularly reduces aphid populations and even epizootics are not rare. The
species spectrum of the pathogenic fungi can be broad but the most frequent and most
effective is the group of four fungal species, which are present each year in the aphid
populations.
5.1.2 DESCRIPTION OF A NEW SPECIES

During a survey of aphidophagous Entomophthorales in Slovakia, the aphid Uroleucon
aeneum (Hille Ris Lambers) colonising Carduus nutans Linné was found being attacked by
fungus with specific taxonomic features allowing us to erect it as a new species. The fungus
fits in almost all respects into the genus Pandora (Humber 1989) by possessing elongate
monokaryotic conidia with an outer wall, which may partially separate from an inner one, and
by having large nuclei staining readily with aceto-orcein. Rather long tapering pseudocystidia
at base 1.5 to twice as thick as conidiophores and branched conidiophores also support the
placement in the genus Pandora. Lack of monohyphal rhizoids would seem to be the only
obstacle in placing the fungus in Pandora, however, there is at least one further valid species
devoid of rhizoids, Pandora delphacis (Hori) Humber, assigned to the genus. Rhizoids are
also fungal structures, which might help readily distinguish Pandora from Erynia or Furia.
In the light of our observations the organism is described here as a new species. The
description that follows is based on a general appearance of the pathogen as it occurs in
naturally infected aphids and on its microanatomy as observed in aceto-orcein (AO).
Pandora uroleuconii Barta and Cagáň, sp. nov.
Diagnosis: Corpora hyphalia elongata, hyphoidea vel irregulariter ramosa, uninucleata
vel oligonucleata (1-6 nucleis praedita), 9.28 – 10.08 (7.92 – 12.87) µm crassa, 120.6 (66.8 -
200.5) µm longa. Pseudocystidia simplicia, 176.6 (138.6 – 207.9) µm longa, ad basim 15.46
(11.88 – 19.80) µm crassa, in medio longitudinis 9.5 (7.92 – 11.88) µm crassa, deinde leniter
attenuata ad 5.99 (5.18 – 6.35) µm in apicibus. Conidiophora ramosa, intertexta in hymenium
99
continuum, 10.18 – 11.33 (9.90 – 12.87) μm crassa, cellulae conidiogenae angustuae in

apicibus in diametro 5.84 – 6.39 (4.95 – 7.72) μm. Conidia primaria uninucleata, bitunicata,
hyalina, elliptica vel ovoidea, dorso-ventraliter asymmetrica, dimensionibus ex insectis 24.71
– 33.03 (21.78 – 41.58) µm x 11.57 – 17.19 (9.90 – 22.77) µm L/D* proportione: 1.79 – 2.25.
Conidia secundaria minora, vel conidia primaria similia (18.14 – 19.21 (15.84 – 27.72) µm x
9.31 – 9.62 (7.92 – 11.88) µm L/D: 1.93 – 2.01) vel fere globosae cum conspicuis papillis
(17.27 – 18.45 (11.88 – 27.72) µm x 9.58 – 10.18 (7.92 – 11.88) µm L/D: 1.70 – 1.93). Nuclei
in corporibus hyphalibus et conidii magni, 5.46 – 7.09 (3.96 – 8.91) µm in diametro. Sporae
perdurantes inobservata. Rhizoidea absunt. Fungus in mediis artificialibus „SDAYEM“,
„EYM“ et „EY“ non crescit. In nymphis et imaginibus Homopterosum (Aphidoidea),
Uroleucon aeneum (Hille Ris Lambers) (hospite typico).
Holotypus: Specimen numero Cr 443 in collectione Instituti Botanici (Slovacum Musaeum
Nationale) designatum, in Komjatice (Slovacia), die 25 mensis Iunii anno 1999, coll. et leg.
M. Barta.
*Abbreviatione L/D utimur, quae in lingua Anglorum proportionem longitudinis ad
crassitudinem significat.
Mycelium develops inside host in a form of mono- to oligokaryotic, simple hyphae-like or

irregularly shaped, sometimes ramified hyphal bodies 9.28 – 10.08 (7.92 – 12.87) µm thick
(4x25 objects) and 120.6 (66.8 – 200.5) µm long (2x25 objects) (Figure 22). Number of
nuclei ranges from 1 to 6 per one hyphal body with average of 2 nuclei (4x25 objects).
Pseudocystidia present but not prominent, simple and narrowing towards the obtuse apex,
15.46 (11.88 – 19.80) µm thick at base (13 objects), 9.5 (7.92 – 11.88) µm (25 objects) thick
in the middle of their length and 5.99 (5.18 – 6.35) µm (25 objects) at the apex, average
length 176.6 (138.6 – 207.9) µm (25 objects). Conidiophores branched interweaving to form a
continuous hymenium more ore less completely covering pleural and dorsal parts of host’s
body, conidiogenous cells slightly enlarged in subapical parts and constricted at their apex
under forming conidia, with a diameter of 10.18 – 11.33 (9.90 – 12.87) µm (2x25 objects) and
5.84 – 6.39 (4.95 – 7.72) µm (2x25 objects) at their constricted necks. Primary conidia
ellipsoid, broadly ellipsoid to ovoid, 24.71 – 33.03 (21.78 – 41.58) µm x 11.57 – 17.19 (9.90
– 22.77) µm L/D: 1.79 – 2.25 (10x25 objects), monokaryotic and bitunicate (with a separable
outer wall layer except over the basal papilla); papilla rounded, centred on spore axis or
displaced laterally. Secondary conidia formed singly on a short conidiophore arising laterally
from a primary conidium and forcibly discharged, more or less similar to primary conidia
18.14 – 19.21 (15.84 – 27.72) µm x 9.31 – 9.62 (7.92 – 11.88) µm L/D: 1.93 – 2.01 (4x25
objects) or broadly ovoid 17.27 – 18.45 (11.88 – 27.72) µm x 9.58 – 10.18 (7.92 – 11.88) µm
L/D: 1.70 – 1.93 (4x25 objects). Nuclei easily distinguished in vegetative or reproductive
structures, staining readily with aceto-orcein and large with diameter of 5.46 – 7.09 (3.96 –
8.91) µm (8x25 objects) in primary spores. Rhizoids unobserved and probably not formed.
Mouthparts and forelegs of cadavers were also carefully examined for rhizoid-like structures,
but no monohyphal structures resembling rhizoids were observed. No discoid-like holdfast
typical to P. neoaphidis was noticed. Resting spores unobserved. The fungus does not grow
on standard media (Sabourad-dextrose agar enriched with milk and egg yolk – SDAYEM, egg
yolk with milk – EYM, or pure egg yolk – EY).
HOLOTYPE: Specimen No. Cr 443, air-dried cadavers of host aphid (exsiccata),
Department of Botany, Slovak National Museum, coll. M. Barta, 25 June 1999, Komjatice,
Slovakia
TYPE HOST: Uroleucon aeneum (Hille Ris Lambers)
TYPE LOCALITY: Komjatice, Slovakia
ETYMOLOGY: Uroleucon – generic name of the type host species
100
__________________________________________________________________________________
Figure 22. Pandora uroleuconii sp. nov.
a) killed aphids adhered to the plant by their proboscises; b) cadaver with released spores around
it; c) hyphal bodies (aceto-orcein “AO”); d) pseudocystidia (psc) protruding over a conidial layer
(AO); e) conidiophores with forming primary conidia; f) primary conidia (left side – AO); g)
secondary conidia (sc) forming on primary conidia (AO).
101
DISTRIBUTION: The species is known only from the type host in Slovakia. The fungus
was observed from 19 localities in the country during late spring and early summer (Table 31,
see appendix), and it is considered a frequent pathogen of the aphid species.
Symptoms of disease: Infected cadavers were easily recognised in aphid colonies by their
conspicuous posture on a host plant and by an obvious change of their colour during fungal
sporulation. With infection progress the mycosed aphids became gradually extended and
turgid but, in general, a shape of aphids’ bodies remained still unchanged. Diseased cadavers
were greyish-brown in colour with a pink tinge immediately after a death. In a final stage of
disease development a conidiogenesis appeared followed by a conidial release under humid
conditions. At sporulation the colour of cadavers changed from greyish-brown to silver. Due
to a lack of rhizoids, aphids killed by the pathogen were attached to the substrate by their
proboscises as well as by means of their forelegs. The attachment of the cadavers was rather
weak and the aphids were easily dislodged from the leaves when being collected. Under dry
conditions the cadavers became quite firm, did not sporulate and remained more or less
mummified. However, the mummies transferred to a humid chamber began to sporulate
readily. The mycelium inside cadavers preserves its viability up to fortnight after storage in
refrigerator and the sporulation started usually about two hours after mummies were put on
damp filter paper.
The infected aphids were for the first time observed on C. nutans in June 1999.
Subsequent investigation of U. aeneum colonies in Slovakia revealed that the fungus was
regular pathogen of the aphid in the course of late spring and early summer, although
epizootic level of mycosis has yet not been observed. Table 31 (see appendix) shows localities
in Slovakia where the pathogen was recorded. According to our observations the “non-
rhizoidal fungus” (described here as P. uroleuconii) was the only pathogen of the aphid
colonies in 1999 and 2001 (In 2000 we did not observe the aphid colonies). On the contrary,
in 2002 besides this fungal species a pathogen with numerous unbranched monohyphal
rhizoids terminating in discoid holdfast was observed within the colonies, as well. The
“rhizoidal fungus” was identified as the most frequent aphidophagous pathogen, Pandora
neoaphidis (Remaudière and Hennebert) Humber. In this year the abundance of P. neoaphidis
cadavers even predominated over that of “non-rhizoidal fungus” in many colonies and in
some of them only P. neoaphidis occurred. Both fungi were easily distinguishable in situ by
external symptoms of mycoses: by a colouration of cadavers and by a characteristic posture of
dead aphids on a host plant associated with presence or absence of rhizoids. As aphids killed
by the “non-rhizoidal fungus” (greyish-brown in colour) were weakly attached to plant with
proboscises, individuals infected by P. neoaphidis (ochre-red in colour) were quite firmly
attached to plant surface by rhizoids emerging from the ventral part of abdomen.
Despite those dissimilarities in external symptoms, the “non-rhizoidal fungus” is
microscopically very similar to P. neoaphidis and other species closely related to P.
neoaphidis, namely P. delphacis. The new species, described here, is however distinguished
from the both Pandora species by growth on standard media and probably high host
specialization. Moreover, it differs from P. neoaphidis by absence of rhizoids and larger
primary conidia (Table 4). Differences in conidial size between those pathogens are probably
of low taxonomical value since the intraspecific variations in the conidial size of the species
are rather great. We assume one source of the intraspecific variation might be a resporulation
of some number of primary conidia deposited on microscopic preparations prepared for
measurement, thus a certain amount of smaller secondary conidia might bias the results.
Anyway, the species cannot be distinguished clearly by the spore size alone, and other
taxonomic characters have to be applied in separating the species. Complete absence of
rhizoids and pathobiology, especially host range or degree of fungal specificity, we consider a
102
Table 4. Gross taxonomical features of the Pandora species

Fungal species P. uroleuconii sp. nov. P. neoaphidis (Rem. and P. delphacis
1
Henn.) Humber 2 (Hori) Humber 3
Primary spore 24.7-33.0 (21.8-41.6) x 21.0-32.0 (15.0-40.0) x 22.9-36.8 x 12.4-
size (μm) LxD; 11.6-17.2 (9.9-22.8); 11.0-14.0 (9.0-16.0); 20.3
L/D 1.8-2.3 1.7-2.3 (1.4-2.9)
Rhizoids absent present absent
no growth on growth on SDAEY, EYM, fast growth on
Culture
SDAYEM, EYM, EY EY 4 SEMA 5
Host spectrum Uroleucon aeneum plenty of aphid species plant- and leaf-
from several families hoppers (aphids
artificially 5,6)
___________________________________________________________________________________________________________________________________________
1
authors’ observations, 2 REMAUDIÈRE and HENNEBERT 1980, 3 HORI 1906, 4 KELLER 1991, 5 MILNER et al.
1983, 6 SHIMAZU 1977
L – length, D – diameter, SDAEY – Sabourad-dextrose-agar enriched with egg yolk, SDAYEM - Sabourad-
dextrose-agar enriched with east extract, egg yolk and milk, SEMA – Sabourad-dextrose-agar enriched with egg
yolk and milk, EYM – egg yolk with milk, EY – egg yolk
much more important taxonomic feature of P. uroleuconii. Taxonomic significance of

presence/absence of rhizoids has been decisively discussed during a development of
nomenclature of Entomophthorales. Some taxonomists, for delimiting entomophthoralean
genera, put no weight on presence of rhizoids in certain species, as they are not always
constant /REMAUDIÈRE and KELLER 1980/, or others regarded a presence of rhizoids to be
taxonomically significant while their absence was not considered a reliable criterion at a
generic level /KING and HUMBER 1981/. Other authorities believe that rhizoids have a value at
subgeneric level /BEN-ZE’EV and KENNETH 1981/ or have value as a secondary generic
character while certain exceptions are accepted, e.g. P. delphacis /HUMBER 1981, 1989/.
BAŁAZY /1993/ questioned the taxonomic significance of rhizoids for P. neoaphidis when he
mentioned that rhizoids of the fungus might be sometimes on particular individuals
underdeveloped or lacking in very dense aphid colonies. Further the author supposed that the
P. neoaphidis, in the sense referred in current literature, is a species complex. This is in
accordance with opinion of HUMBER /1983/. Certain morphological and physiological
variation among P. neoaphidis strains was highlighted by KELLER /1991/, as well. We assume
that instability in presence of rhizoids observed by BAŁAZY /1993/ for P. neoaphidis could be
one of signs of variability inside of the taxon. During our investigation the absence of rhizoids
was unquestionably a constant character of P. uroleuconii as it was noticed on all cadavers in
different localities all over the country during four consecutive years. This character was also
occurred on cadavers of U. aeneum artificially inoculated by the pathogen in the laboratory.
The absence of rhizoids was, moreover, associated with other physiological properties of the
taxon what differentiated it from “rhizoidal” P. neoaphidis.
Besides morphological features discussed before, P. uroleuconii is close to P. delphacis by
absence of rhizoids. However, both species differ in host specificity and growth in artificial
cultures. P. delphacis is primarily a pathogen of leaf- and planthoppers in Asian rice paddies
/HORI 1906, HOLDOM et al. 1989, MATSUI et al. 1998/, although the pathogen artificially
infected some aphid species in laboratory /SHIMAZU 1977, MILNER et al. 1983, XU and FENG
2002/. The pathogen exhibited even greater pathogenicity to aphids than P. neoaphidis /XU
and FENG 2000/, however it has never been observed infecting aphids in nature and what is
more the species has never been recorded from Europe. In our experiments all attempts for an
artificial transmission of P. uroleuconii to five aphid species, Acyrthosiphon pisum Harris,
Diuraphis noxia (Mordvilko), Metopolophium dirhodum (Walker), Myzus persicae (Sulzer)
103
and Rhopalosiphum padi (Linné), were unsuccessful. The pathogen infected only the type
host, thus indicating what is probably a very narrow host specialization. P. delphacis grows
more rapidly in vitro than P. neoaphidis and even it grows on a wider variety of media
(nutritionally simple and complex) /HUMBER 1981, MILNER et al. 1983/. P. uroleuconii
unlike those two species did not grow on artificial media. Any attempts to isolate the species
failed. Spores of the pathogen apparently germinated after their deposition on media but a
hyphal growth ceased shortly; this observation may support our supposition about pathogen’s
high specificity. Two strains of P. neoaphidis were, however, isolated on SDAYEM from
killed U. aeneum, and the strains showed pathogenicity to the five aphid species mentioned
above. The inability to gain isolates of P. uroleuconii prevented us from testing the
biochemical and molecular properties that might provide the clearest separation of those
morphologically similar species. However, recent progress in methods for recovery of fungal
DNA from insect cadavers to conduct molecular studies /Nielsen et al. 2003/ gives hope for
early solution for the taxonomic problem.
P. uroleuconii has been defined by qualitative description of the external morphology,
quantitative measurements of the main taxonomical structures as well as by observations on
host preferences. Although the taxon shows a morphological resemblance in certain features
to some representatives of Pandora genus, the results of examination presented here we
regard to be substantial enough to justify the erection of the new species.
5.1.3 A KEY TO APHID PATHOGENIC ENTOMOPHTHORALES

The key to entomophthoralean species parasiting aphids presented herein is a result of our
endeavour to assemble any information from literature relating to the taxonomy of aphid
pathogenic Entomophthorales. A considerable progress in the knowledge on the taxonomy of
Entomophthorales has been made in the last decade and a plenty of literature concerning the
aphid pathogenic Entomophthorales has been published. Unfortunately the publications are
dealing with species of specific genus as a whole or the order at generic levels and papers
including all the aphid-pathogenic species of this particular group of fungi have been still
missing. This key is mainly a synthesis of the most influential taxonomic monographs on
Entomophthorales published recently /HUMBER 1990, KELLER 1987a, KELLER 1991, BAŁAZY
1993, KELLER 1997, 1999, 2002/ and some fresh data were added.
1a Only spherical resting spores known.……………… ... Tarichium – a form genus 10

1b Conidia present…………………………………………………………………... 2
2a Primary conidia spherical, uninucleate and unitunicate; nuclei large, greater than
5 µm in diameter, staining weakly in AO*; conidiophores unbranched strongly
inflated in subapical part; secondary conidia like primary, capilliconidia or
microconidia; saprophytic species on different organic matter, occasionally
pathogens of insects or vertebrates……….... Basidiobolus (fam. Basidiobolaceae) 11
2b Primary conidia uninucleate or multinucleate, but if uninucleate then bitunicate
with large nuclei staining readily with AO and conidiophores branched; if
multinucleate then unitunicate with small to medium nuclei well or not staining
with AO and conidiophores unbranched……………………………………………. 3
3a Primary conidia unitunicate with more than one nucleus, staining well or not in
AO, but staining well in FRS*; primary conidia bell-shaped, spherical, pyriform or
broadly ellipsoidal in shape; conidiophores unbranched; rhizoids present or absent
and pseudocystidia absent…………………………………………………………... 4
104
3b Primary conidia uninucleate and bitunicate; nuclei relatively large and staining
readily with AO; primary conidia elongate pyriform, fusiform or cylindrical in
shape; conidiophores branched; rhizoids and pseudocystidia usually present……... 8
4a Primary conidia typically bell-shaped with apical point (Lakon’s type: truncata-
campaniformis); projected primary conidia surrounded by ruptured conidial wall;
conidia with 2 to 20 (30) nuclei staining well in AO; secondary conidia similar to
primary ones but without the apical point..…………………………………….……
………………………………………...Entomophthora (fam. Entomophthoraceae) 12
4b Primary conidia spherical to pyriform with≥ 4 nuclei, without api cal point and
not surrounded by ruptured conidial wall…………………………………………... 5
5a Primary conidia relatively small, spherical with truncate papilla, smoky spore
wall and with 4-8 nuclei not staining with AO; secondary conidia like primary or
amygdaliform capilliconidia on long distally bent capillary; resting spores
ellipsoidal with black (black-brown) episporium...Neozygites (fam. Neozygitaceae) 13
5b Primary conidia relatively large, subspherical to pyriform with more than 8
nuclei, papilla obtuse or pointed……………………………………………………. 6
6a Nuclei small, not or just weakly staining with AO, on average more than 50
nuclei per conidium; secondary conidia like primary ones, or microconidia, or
capilliconidia produced (not in aphidophagous species), weak rhizoids rarely
present. ..………………………………….……Conidiobolus (fam. Ancylistaceae) 18
6b Nuclei large, deeply staining with AO; secondary conidia like primary……….… 7
7a Hyphal bodies spherical to subspherical or elongated; nuclei deeply staining with

AO and 4-6 µm in diameter; conidiophores clavate without neck-like narrowing
part or only with very short one (see 7b); rhizoids absent……………….
…………………………………………..Entomophaga (fam. Entomophthoraceae) 23
7b Hyphal bodies irregular, rounded to amoeboid; nuclei deeply staining with AO,
3-5μm in diameter; conidiophores simple, distended in apical part, gently
narrowed and elongated into relatively long neck; rhizoids present or absent, if
present then monohyphal, ended with disc-like holdfast. …………………………..
……………………………………………….…Batkoa (fam. Entomophthoraceae) 24
8a Primary conidia fusiform or cylindrical; papilla conical, pointed or rounded,

demarcated from conidial body by a collar; secondary conidia like primary ones or
sickle- to crescent-shaped on long, slender capillaries; rhizoids pseudorhizomorphs
…………………………………………….Zoophthora (fam. Entomophthoraceae) 25
8b Primary conidia pyriform, fusiform or subcylindrical; papilla smoothly rounded,
not clearly demarcated from conidial body; secondary conidia like primary ones or
spherical with or without small apical point; rhizoids monohyphal………………... 9
9a Pseudocystidia distinctly two to several times thicker than conidiophores, usually

long and often clavate, nodular or furcate at tips; secondary conidia like primary
ones or spherical, and sometimes tetraradiate (in water habitat); rhizoids at least
twice as thick as conidiophores without distinct terminal holdfast……………..…..
……………………………………………….…Erynia (fam. Entomophthoraceae) 31
105
9b Pseudocystidia little thicker than conidiophores and tapering apically;

tetraradiate secondary conidia not present; rhizoids (may be absent) ribbon-like 2-
3 times thicker than conidiophores terminating in discoid or irregularly branched
and spreading holdfast……………………......Pandora (fam. Entomophthoraceae) 32
10 Only dark brown-black spherical resting spores with diameter of 38-45 µm are
present inside of dead aphids; episporium is covered with stubby conical spines….
……………………………………………………………. Tarichium atrospermum
11 Conidiophores 300-400 µm or more in length, 4-7 µm thick, at subapical part 12-

15 µm thick; primary spores 23-48 µm in diameter; capilliconidia 65-85 x 21-24
µm; resting spores 31-38 µm in diameter………………….. Basidiobolus ranarum
12a Primary conidia more than 15 µm in length; primary conidia on average 14-23 x
12-19 µm /MACLEOD et al. 1976/ [15.6-21.4 x 13.4-18.3 µm]*; aphidophagous
species with distribution in Europe and other parts of the world.……………..……
……………………………………………………... Entomophthora planchoniana
12b Primary conidia less than 15 µm in length; primary conidia on average 14.4-12.3
µm /HUMBER and FENG 1991/; aphidophagous species with distribution outside of
Europe……………………………………………….Entomophthora chromaphidis
13a On aphids…………………………………………………………………………. 14
13b On other Homoptera………………………………………………………………. 17
14a On Aphidinae……………………………………………………………………... 15
14b On Lachninae, Pterocommatinae, or Phyllaphidinae……………………………... 16
15a Hyphal bodies spherical to subspherical, 14-17 µm; primary conidia 18-22 x 14-
18 µm [17-21 x 14-17 µm, L/D*= 1.1-1.5], predominantly with 4 nuclei;
capilliconidia 20-27 x 11-14 µm, L/D= 1.5-2.4 [19.2-30.1 x 10-17 µm, L/D= 1.4-
2.5]; capillary tube on average 17-35 µm [20-66 µm]; resting spores ellipsoid 30-
41 x 18-23 µm [41-43 x 21-22]; mainly on Aphis spp., but also on other genera
/KELLER 1997/.…………………………………...……………..Neozygites fresenii
15b Hyphal bodies spherical to subspherical, 17-22 µm; primary conidia 24-26 x 18-
19 µm [35.6-36.2 x 29-30 µm, L/D= 1.2], predominantly with 5 nuclei;
capilliconidia 30-34 x 12-15 µm, L/D= 2.3-2.6 [26-30 x 11-14 µm, L/D= 2.1-2.3];
capillary tube on average 150-170 µm [96-108 µm]; resting spores ellipsoid 35-43
x 20-23 µm; on Microlophium spp. /KELLER 1997/...............Neozygites microlophii
15c Primary conidia 28-32 x 17-22 µm; capilliconidia 29-35 x 16-18 µm; on
Myzocallis coryli (Goetze) and unidentified species /KELLER 1997/...................…..
...........................................................................................Neozygites lageniformis
16a Hyphal bodies spherical to subspherical, 15-24 µm; primary conidia 19-23 x 12-
17 µm with 8 nuclei on average; resting spores ellipsoid 32-35 x 21-22 µm [38 x
23 µm, L/D= 1.6-1.7]; [germ capilliconidia 33.3-33.6 µm, capillary tube on
average 73 µm]; on Pterochloroides persicae (Cholodkovskii), Tuberolachnus
salignus (Gmelin) /KELLER 1997/ and Pterocomma salicis (Linnaeus) [in this
work]...……………………………………………………..….Neozygites turbinata
106
16b Hyphal bodies spherical 20-22 µm; primary conidia 24-31 x 18-21 µm [24-27 x
14-15 µm, L/D= 1.7], predominantly with 4 nuclei; capilliconidia 32-34 x 14-17
µm, L/D= 2.0-2.5 [32-34 x 14-16 µm, L/D= 2.1-2.2]; resting spores ellipsoid 34-
36 x 23-24 µm [32-24 x 22-23 µm, L/D= 1.5]; on Cinara pilicornis (Hartig)
/KELLER 1997/…...…………………………………………….. Neozygites cinarae
17 On Lecaniidae. Hyphal bodies spherical; primary conidia 18 x 9-10 µm;

capilliconidia 12-20 x 5-10 µm; resting spores subspherical to broad ovoid, dark;
on Lecanium viridae Green (HUMBER /1990/ states the species as a pathogen of
aphids)………………….…………………………………..…… Neozygites lecanii
18a Multiplicative resporulation by microconidia present; villose resting spores……. 19

18b Microconidia absent; resting spores with more or less smooth walls…………….. 20
19 Primary conidia on average 38-40 x 50-53 µm; resting spores 30-32 µm on

average /KELLER 1987a/; microconidia 15 x 11 µm /PRASERTPHON 1963/; on
different insect host and detritus, occasionally on vertebrates………………………
……………………………………………………………..Conidiobolus coronatus
20a On hibernating mosquitoes (on aphids artificially); primary conidia 22.5-31.0 x

18.0-24.0 µm; rhizoids present /WEISER and BATKO 1966/………………………...
……………………………………………………………...Conidiobolus destruens
20b On aphids or other insects; rhizoids absent……………………………………….. 21
21a On Phytonomus spp. larvae or aphids; primary conidia on average 25-37 x 22-30
µm; resting spores 13-37 µm in diameter; in cultures characteristic odour of
benzene hexachloride /BAŁAZY 1993/……….……………...Conidiobolus osmodes
21b On aphids…………………………………………………………………………. 22
22a Primary conidia on average 33.5-44 x 28-36 µm, L/D= 1.2-1.3 [31.1-42.5 x 26.0-
36.8 µm, L/D= 1.1-1.2]; resting spores 34-38 µm [32.2-38.5µm] in diameter
/KELLER 1987/…..…………………………………………..Conidiobolus obscurus
22b Primary conidia 24-32 x 17.5-26.5 µm [24.4-27.2 x 18.6-21.0 µm, L/D= 1.3] on
average; resting spores 15-27 µm in diameter /BAŁAZY 1993/…………….………
…………………………………………………………..Conidiobolus thromboides
23 Primary conidia pyriform 15-31 x 12-25 µm, L/D= 1.17-1.50, with markedly
long papillar neck; resting spores 12-25 µm in diameter /THOIZON 1967/….……
………………………………………………………..…..Entomophaga pyriformis
24a Primary conidia together with papillae 48-52 (57) x 40-47 µm /BAŁAZY 1993/;
usually on beetles, hymenopterans and crane flies. …. …………….. Batkoa major
24b Primary conidia together with papillae 30-38 x 29-34 µm /BAŁAZY 1993/; mostly
on planthoppers and flies………………………………………….Batkoa apiculata
25a Primary conidia of two types: cylindrical elongate with rounded ends of L/D
ratio about 3, and ovoid or short ellipsoid, distinctly thicker of L/D ratio 2 or less
than 2; capilliconidia unknown……………………………………………………... 26
107
25b Primary conidia of one type, slender ellipsoid or cylindrical of L/D always more
than 2; capilliconidia observed……………………………………………………... 27
26 Primary conidia ovoid to short ellipsoid 12.6-30.8 x 8.2-16.5 µm, L/D= 2 or long
ellipsoid 17.1-33.3 x 5.9-12.9 µm, L/D= 3; resting spores 22-32 µm in diameter,
exogenously produced; on Myzus persicae Sulzer in China /LI 1986/……………...
……………………………………………….……………..Zoophthora anhuiensis
27a Primary conidia 15-22 x 5.5-7.5 µm on average, L/D= 2.4-3.0 [14.9-19.2 x 5.9-
7.3 µm, L/D= 2.5-2.6]; capilliconidia 13-22 x 5-7 µm, L/D= 2.7-3.7 [18.1-18.9 x
5.1-5.6 µm, L/D= 3.4-3.6]; resting spores 24-29 µm; on aphids and other hosts
/REMAUDIÈRE and HENNEBERT 1980/..…………..……….…..Zoophthora radicans
27b Primary conidia longer than 20 µm on average, their average length is usually
above 25-35 µm, conidia shorter than 20 µm do not occur or sporadically……….. 28
28a Length of primary conidia seldom exceeds 30 µm, their average length is closed
in the range of 25-30 µm, and average L/D ratio is less than 3.5…………………... 29
28b Primary conidia commonly longer than 30 µm, their average length is closed in
the range of 30-40 µm, and average L/D ratio is over 3.5………...………………... 30
29a Primary conidia 27-32 x 9-12 µm, L/D= 2,5-3,2 [29.0-30.1 x 12.9-15.1µm, L/D=
2.0-2.3]; capilliconidia amygdalyform 20-23 x 10-12 µm, L/D= 1.9-2.2 [28.3-29.3
x 12.7-13.4 µm, L/D= 2.2]; resting spores 37-40 µm [37.1-41.3 µm]; on Anoecia
spp. or other aphids /REMAUDIÈRE and HENNEBERT 1980/….....Zoophthora aphidis
29b Primary conidia 23.7-34.8 x 6.3-10.3 µm, L/D= 3,2-3.3; capilliconidia like plum
fruit in shape 24.5-29.2 x 5.5-10.3 µm; capillary tubes 50-60 µm long; resting
spores unobserved; on Aphis citricola v.d. Goot in Israel /BEN-ZE’EV and
KENNETH 1981/………………………………………….….. Zoophthora orientalis
29c Primary conidia 15-36.5 x 6.5-14.5 µm, L/D= 2.5; capilliconidia strongly
elongate somewhat crescent-shaped 33 x 9 µm; resting spores 29-43 µm in
diameter; on Schizolachnus piniradiatae (Davidson) in Canada /MACLEOD et al.
1979/………………………………………………………..Zoophthora canadensis
30a Primary conidia 32-48 x 11-14 µm; capilliconidia 18 x 7 µm; capillary tubes 80-
120 µm long; resting spores unknown; on different aphid species /BAŁAZY 1993/;
primary conidia cylindrical with parallel sides in outline, hemispherically convex
papilla and broadly obtuse ends /MIETKIEWSKI et al. 1981/...Zoophthora phalloides
30b Primary conidia 30-35 x 7-8.5 µm, L/D= 4.2; capilliconidia almond-shaped 17-
25 x 6-7.7 µm; capillary tubes 55-85 µm long; resting spores 24-30 µm in
diameter; on several aphid species /BAŁAZY 1993/; primary conidia ellipsoidally
spindle-shaped with sides a little convex, papilla widely conical or slightly convex
often with small sharp central apiculus, top part of the spore tapering with
somewhat blunt apex /MIETKIEWSKI et al. 1981/..…...….. Zoophthora occidentalis
31a On aphids; primary conidia on average 12.6-20.5 x 7.1-13.4 µm, L/D= 1.4-2.2;
echinulate resting spores 27.7-37.1 µm [30.7-32.1 µm] in diameter /BEN-ZE’EV
and KENNETH 1979/...…………………………………..……….…Erynia erinacea
108
5. RESULTS AND DISCUSSION: 5.2 Biological characteristics of Pandora neoaphidis isolates
31b Mostly on Culicidae and Chironomidae (observed from aphids /THOIZON 1970/);
primary conidia 27-80 x 12-14 µm; tetraradiate secondary conidia can be
produced; resting spores 30-50 µm in diameter /BAŁAZY 1993/….…Erynia conica
32a Rhizoids absent…………………………………………………………………… 33

32b Monohyphal rhizoids present……………………………………………………... 34
33a Primary conidia on average 26.6 x 15.5 µm; resting spores 24-28 µm in diameter
(from aphid host /SHIMAZU 1977/); pathogen of plant- and leafhoppers, on aphids
artificially………………………………….……………………Pandora delphacis
33b Primary conidia on average 24.7-33.0 x 11.6-17.2 µm, L/D= 1.8-2.3; resting
spores unobserved; no growth on Sabouraud’s agar; pathogen of Uroleucon
aeneum (Hille Ris Lambers) in Slovakia……………………...Pandora uroleuconii
34a Resting spores present; primary spores less than 18 µm in length; primary spores
15-16.6 x 8-10.5 µm [17 x 11 µm, L/D= 1.6]; resting spores 24.9-30.4 µm [26-27
µm] /REMAUDIÈRE and HENNEBERT 1980/; on Pemphigus bursarius Linnaeus,
Aphis fabae Scopoli, Aphis umbrella Börner /REMAUDIÈRE and HENNEBERT 1980/,
Aphis acetosae Linnaeus [in this work]…….………………...……Pandora nouryi
34b Resting spore unknown; primary spores more than 18 µm in length…………….. 35
35a Primary conidia 21-32 x 11-14 µm, L/D= 1.7-2.3 [21.5-28.1 x 10.8-14.5, L/D=
1.6-2.2 µm] /REMAUDIÈRE and HENNEBERT 1980/; on various aphid species……
………………………………………………………………....Pandora neoaphidis
35b Primary conidia 16-21 x 10x12 µm; morphologically similar to P. neoaphidis,
but hyphal bodies more branched up to 200 µm long, it also differs biochemically
by means of fatty acid composition (mycelium contains 21-46% C12:0 in contrast,
P. neoaphidis contains only 3-6% C12:0), or isoenzyme patterns /MILNER et al.
1983/; pathogen of aphids in Australia and China……….…...Pandora kondoiensis
__________________________
* AO- aceto-orcein stain; FRS- Feulgen reaction stain; L/D-length/diameter ratio of conidia; [our measurements]
5.2 BIOLOGICAL CHARACTERISTICS OF PANDORA NEOAPHIDIS ISOLATES

The entomopathogenic fungus P. neoaphidis has been often considered the most important
natural regulator of aphid colonies /PELL et al. 2001/ and has long been investigated as a
potential biological control agent. Augmentation of P. neoaphidis in field and greenhouse
bioassays has been attempted but with limited success /WILDING 1981, WILDING et al. 1990,
SHAH et al. 2000a/. In general, a strain with high virulence is necessary to select for
inoculative or inundative augmentation biocontrol strategies /PELL et al. 2001, SHAH and PELL
2003/. Conservation biocontrol strategies rely not on inoculum release but on the providing of
favourable conditions within agroecosystems to enhance entomopathogen activity
/EILENBERG 2002/. For conservation biocontrol it is likely that a diverge range of fungal
strains need to be encouraged and these strains should be able to infect target and alternative
hosts /PELL et al. 2001/. Recently, a three tiered assay system has been devised for assessment
of P. neoaphidis isolates against aphids within the programme of biological control /Shah et
al. 2004/. Both augmentation and conservation programmes require information on
intraspecific variability in pathogen population since the variability of fungal strains in
agroecosystems may enhance its impact.
109
15 isolates of the entomophthoralean fungus P. neoaphidis, isolated from different hosts

and localities in Slovakia (Table 25, see appendix), were tested to evaluate an intraspecific
variation within the taxon. The small group of Slovak isolates was obtained from fungus-
killed aphids collected in various part of the country, mostly in the south-western Slovakia.
Eight isolates were originally obtained from pest aphid species and seven isolates were from
non-pest aphids. Majority of the isolates have a different origin, but there are also isolates
obtained from aphid cadavers collected in the same date during epizootics. This allows us to
make insight into variability of the fungal species acting within host populations even if
isolated from the same host aphid collected at the same site. The study concentrates on
analysis of phenotypic characters of the species, including a morphology of primary conidia, a
conidium germination on different substrates, a virulence against aphid, a radial growth of
isolates, and a biomass production.
5.2.1 SIZE OF PRIMARY CONIDIA

Dimensions of primary spores of P. neoaphidis isolates are presented in the Table 5. Two
morphological characteristics, the conidial length (L) and the conidial diameter (D), were
measured and a ratio of the length to the diameter was calculated. The dimensions varied
among the isolates indicating a great heterogeneity. A one-way ANOVA showed significant
difference among characteristics of primary spores at 99% confidence level (F 2.51 = 76.35, P >
0.01 for length; F 2.51 = 57.30, P > 0.01 for diameter; and F 2.51 = 40.45, P > 0.01 for the ratio
of L/D). A multiple comparison procedure to determine which values are significantly
different from which others revealed 9 homogenous groups for the length and the L/D ratio,
Table 5. Dimensions of primary conidia of the P. neoaphidis isolates
Isolate Length (L) (µm)* Diameter (D) (µm)* Ratio L/D

1 35.60 – 37.38 (29.70 – 43.56) i** 17.70 – 18.14 (14.85 – 21.75) ef** 2.01 – 2.06 ef**
2 31.09 – 32.47 (25.74 – 37.62) fg 16.04 – 16.51 (13.86 – 21.78) d 1.88 – 2.02 de
3 35.20 – 35.80 (27.72 – 43.56) i 18.93 – 20.59 (15.84 – 25.74) g 1.72 – 1.86 bc
4 26.77 – 30.81 (23.76 – 35.64) bc 16.28 – 20.08 (13.86 – 23.76) f 1.53 – 1.67 a
5 24.16 – 25.54 (20.79 – 32.67) a 11.48 – 12.24 (9.90 – 17.82) a 2.08 – 2.10 fgh
6 30.89 – 31.72 (25.74 – 35.64) ef 14.34 – 14.93 (11.88 – 19.80) c 2.12 – 2.21 h
7 29.74 – 31.44 (25.74 – 37.62) de 16.55 – 17.62 (13.86 – 19.80) d 1.78 – 1.87 c
8 29.86 – 31.40 (23.76 – 39.60) de 13.58 – 15.84 (10.89 – 17.82) bc 1.93 – 2.30 gh
9 28.99 – 29.94 (25.74 – 35.64) cd 13.86 – 14.22 (11.88 – 17.82) bc 2.09 – 2.12 fgh
13 23.21 – 25.86 (19.80 – 35.64) a 12.12 – 14.18 (9.90 – 17.82) b 1.77 – 1.92 c
14 31.56 – 34.21 (25.74 – 41.58) gh 16.32 – 18.18 (13.86 – 25.74) de 1.88 – 1.99 d
16 35.09 – 36.55 (29.70 – 53.46) i 17.66 – 18.93 (13.86 – 21.78) f 1.93 – 2.00 de
17 32.33 – 34.06 (25.74 – 45.54) h 18.73 – 20.16 (13.86 – 23.76) g 1.69 – 1.75 b
18 27.84 – 28.23 (23.76 – 35.64) b 11.72 – 12.71 (9.90 – 15.84) a 2.22 – 2.38 i
19 29.66 – 29.74 (25.74 – 37.62) cd 14.34 – 14.49 (11.88 – 17.82) c 2.05 – 2.07 fg
* Dimensions consist of two ranges: the range of the extreme mean values of the 4 series (one series = 25
objects) and the range of extreme values of the measurement (in parentheses)
** Means followed by the same letter within each column are not significantly different (Tukey’s HSD test,
P<0.05)
110
and 7 homogenous groups for the diameter. These results confirmed a great biometrical
heterogeneity among the isolates. Conidial size of isolates, which originated from the same
host, site, and date (isolates of numbers 6, 7 and 8, 9) were not significantly different.
The dimensions of conidia produced in vitro generally exceeded the ranges for in vivo
conidia described by KELLER /1991/ 22.7 – 25.0 µm for length and 8.5 – 15.0 µm for
diameter, or the ranges by BAŁAZY /1993/ 21.0 – 32.0 x 11.0 – 14.0 µm. However, the L/D
ratios corresponded with that published by KELLER /1991/. In other studies on intraspecific
variation in P. neoaphidis strains the differences in conidial size were examined and very little
variation was found /MILNER et al. 1983/. The authors also observed that primary conidia
from cultures were obviously larger than those from insects. This is in agreement with
MORGAN /1994/ who found that in vitro-produced conidia of P. neoaphidis were significantly
larger than in vivo-produced conidia. Likewise, we also found that in vivo-produced conidia
are significantly smaller (F 7.64 = 45.19, P > 0.01 for length; F 7.64 = 21.52, P > 0.01 for
diameter). Dimensions of in vivo-primary spores measured from killed aphids prior the
isolation are in the Table 6. Measurements of the in vivo-produced conidia are consistent with
those presented by KELLER /1991/ and BAŁAZY /1993/. However, certain degree of variability
was detected in biometrics of conidia collected from aphid cadavers, as well (Table 6) (F 2.04 =
2.69, P > 0.05 for length; F 2.04 = 29.62, P > 0.05 for diameter; and F 2.04 = 7.45, P > 0.05 for
the ratio of L/D). A variation in morphology is typical for the species /REMAUDIÈRE and
HENNEBERT 1980, KELLER 1991, BAŁAZY 1993/ even it is considered a species complex
/HUMBER 1990, BAŁAZY 1993/.
Table 6. Dimensions of in vivo-primary conidia of the P. neoaphidis isolates collected from aphid
cadavers prior to the isolation
Isolate Length (L) (µm)* Diameter (D) (µm)* Ratio L/D

1 25.60 – 27.72 (20.79 – 29.43) c** 12.55 – 13.46 (11.88 – 17.82) ef** 2.04 – 2.06 de**
2 22.09 – 24.80 (20.79 – 25.74) ab 11.45 – 12.16 (9.90 – 13.86) bc 1.93 – 2.04 cd
3 24.24 – 25.08 (21.78 – 25.74) abc 13.62 – 13.85 (10.89 – 15.84) g 1.78 – 1.81 ab
4 21.97 – 23.65 (19.80 – 23.76) a 14.17 – 14.95 (10.89 – 17.82) h 1.55 – 1.78 a
5 21.16 – 24.32 (20.79 – 25.74) a 10.96 – 11.81 (9.90 – 15.84) ab 1.93 – 2.06 cd
6 23.21 – 25.86 (19.80 – 29.70) abc 11.49 – 12.20 (9.90 – 15.84) bc 2.02 – 2.12 dc
7 22.45 – 26.64 (21.78 – 29.70) abc 11.63 – 12.00 (10.89 – 17.82) bc 1.93 – 2.22 de
8 21.12 – 24.45 (19.80 – 25.74) a 10.67 – 11.53 (9.90 – 15.84) a 1.98 – 2.12 de
9 20.98 – 23.75 (17.82 – 25.74) a 10.60 – 11.10 (9.90 – 17.82) a 1.98 – 2.14 de
13 22.99 – 24.32 (19.80 – 35.64) ab 12.16 – 12.91 (11.88 – 15.84) de 1.78 – 2.00 bc
14 24.32 – 27.18 (21.78 – 30.25) bc 13.74 – 14.08 (10.89 – 17.82) g 1.77 – 1.93 bc
16 24.12 – 27.99 (21.78 – 29.70) c 12.83 – 14.07 (11.88 – 17.82) fg 1.88 – 1.99 cd
17 23.87 – 27.56 (20.79 – 30.25) bc 13.86 – 14.05 (9.90 – 17.82) g 1.75 – 1.99 bc
18 22.44 – 24.75 (19.80 – 29.70) ab 10.74 – 11.88 (9.90 – 15.84) ab 2.09 – 2.30 d
19 23.73 – 27.18 (19.80 – 30.25) bc 11.87 – 12.64 (10.89 – 17.82) cd 2.00 – 2.15 de
* Dimensions consist of two ranges: the range of the extreme mean values of the 4 series (one series = 25
objects) and the range of extreme values of the measurement (in parentheses)
** Means followed by the same letter within each column are not significantly different (Tukey’s HSD test,
P<0.05)
111
5.2.2 VIRULENCE OF THE ISOLATES

The measure of virulence is usually obtained by means of bioassay methods, in which
group of test-insects are submitted to various concentrations of infective conidia. The results
are expressed in terms of regression line of probit transformed mortality and log
concentration. Virulence is usually quantified as the spore concentration causing 50%
mortality in a test population /FINNEY 1971/. HUBER and HUGHES /1984/ discussed the use of
the exponential or independent action model as an alternative to the probit analysis. For
entomophthoralean fungi the bioassay procedures using sporulating cadavers or in vitro
cultures as an inoculum source have been described in the number of works /e.g. WILDING
1976, PAPIEROK 1982, OGER and LATTEUR 1985, LATGÉ and PAPIEROK 1988, PAPIEROK
and HAJEK 1997/. Estimates of lethal concentrations (LC 50 ) of conidia of Slovak P.
neoaphidis isolates for the pea aphid, A. pisum, are presented in the Table 7. The estimated
LC 50 values ranged from 24 to 214 conidia per mm2. This indicates a rather great variability
among the isolates. A one-way ANOVA showed significant differences in the LC 50 values
among the isolates at 99% confidence level (F 2.74 = 4.91, P > 0.01). Multiple range tests
allowed us to identify 4 homogenous groups at the confidence level of 95%. Overall, average
virulence for all the Slovak isolates was 138.35 conidia per mm2. Several previous studies
have reported LC 50 values that are much smaller than the ones presented in this work /e.g.
MILNER and BOURNE 1983, FENG and JOHNSON 1991, SIVČEV and DRAGANIĆ 1994,
SIEROTZKI et al. 2000, SHAH et al. 2004/. Usually, values of LC 50 range from a few conidia to
tens conidia per mm2 /PAPIEROK 1982/. SIEROTZKI et al. /2000/ state a value of 16 conidia per
mm2 as a mean LC 50 for 4 Swiss isolates tested. Out of the Slovak isolates only 4 ones were
highly virulent to pea aphid with virulence below a level of 50 conidia per mm2. On the
contrary 6 isolates had the estimated virulence about 200 conidia per mm2. The relatively low
virulence of the isolates could be attributed to the fact that neither of the isolates was
originally obtained from A. pisum, the aphid used in the bioassay. However, SIEROTZKI et al.
/2000/ found no significant difference in virulence among strains isolated from aphid species
different or the same to the species used in bioassay. On the contrary, SHAH et al. 2004
presented results where isolate of P. neoaphidis obtained from M. persicae was approximately
3-10 times more virulent to A. pisum than to its original host. The pea aphid was, in general,
the most susceptible to P. neoaphidis isolates tested irrespective of isolate origin. It is difficult
to make direct comparisons among the LC 50 values presented in this study and previously
published works, because of differences in bioassay methods, fungal isolates and aphid
species. A part of results on P. neoaphidis virulence published in the literature was based on
exploitation of conidia produced directly from the aphid cadavers. It is known that conidia
produced in vivo are more infective than conidia projected by in vitro cultures. The ratio of
“in vitro” LC 50 values to “in vivo” LC 50 values of the same strain of C. obscurus was put to
ratio of 1:3 /PAPIEROK 1982/. The relatively low virulence in our study could also be a result
of using nymphs in the bioassay. Nymphs can be less susceptible to the pathogens than adults
because the invading conidia are shed with the old cuticle on ecdysis /LATGÉ and PAPIEROK
1988/. We did not use adults in the bioassay because a great part of them died of non-
infection reasons before they could be infected with fungus. Virulence of isolates may
decrease significantly during long-term maintenance in vitro /PAPIEROK 1982, MILNER et al.
1983/. Each isolate used in our experiments were passaged through specimen of pea aphid and
re-isolated not longer than 6 months prior to the bioassay.
A great variation in virulence among the isolates is obvious. We even determined
significant differences among isolates obtained from the same site and aphid species in the
same time. This is for example the case of isolates of numbers 1,2, and 3 isolated from M.
persicae during fungal epizootic. The fungus-killed aphids even originated from the same
tobacco plant. A significant difference was found out between isolates 1 and 2. Similarly,
112
virulence was significantly different between isolates 5 and 7 obtained from M. carnosum,
and isolates 8 and 9 obtained from a single colony of S. avenae on a wheat ear. Apparent
differences between isolates originating from the same host and collection site was detected
using molecular analysis by SIEROTZKI et al. /2000/ and ROHEL et al. /1997/. Some degree of
variation can be observed between various values of LC 50 for one strain with one test-insect
species evaluated at different times /PAPIEROK 1982, PAPIEROK and HAJEK 1997/. OGER and
LATTEUR /1985/ demonstrated that 5 doses, 10 experimental aphids, 8 repetitions, i.e. 400
aphids in total, appeared to be the most efficient for statistically distinguishing two values of
LC 50 with a ratio of 1-2. In our experiments we used 5 doses, 20 aphids and 3 repetitions. A
source of variability in the results can also come from heterogeneity in the groups of aphids
submitted to given amounts of inoculum. The degree of resistance of aphids to the fungi can
depend on numerous parameters, e.g. developmental stage and physiological state of the
insects /LATGÉ and PAPIEROK 1988/. As presented in the “ Materials and Methods” (section
4.3.5) we tried to obtain test aphids as homogenous as possible by using a clone of single
mother aphid, the same larval instar, and a maximal standardization of rearing conditions.
The most frequently published values for the slopes of regression lines range from 1 to 2
/PAPIEROK and HAJEK 1997/. According to PAPIEROK /1982/ extreme values of the slopes are
0.8 and 3.3, while the most frequent values range from 1 to 2.5. In our analyses the extremes
of the slopes were 0.84 and 2.19, but the most of the values ranged from 1 to 2.
Table 7. LC 50 values for the isolates of P. neoaphidis against Acyrthosiphon pisum
Number of isolate LC 50 (conidia/mm2) 95% confidence limits Slope of reg. ± SE**

I. 31.35 19.90 – 49.40 1.44 ± 0.12
1 II. 17.55 6.30 – 49.00 0.89 ± 0.21
III. 22.55 10.70 – 47.65 1.54 ± 0.21
Mean 23.82 a*
I. 328.30 153.00 – 704.30 1.33 ± 0,20
2 II. 127.20 46.50 – 208.80 1.47 ± 0.13
III. 153.05 99.75 – 234.80 1.39 ± 0.11
Mean 202.85 cd
I. 37.2 24.00 – 57.75 1.65 ± 0.13
3 II. 64.65 38.45 – 108.80 1.16 ± 0.12
III. 78.00 56.05 – 108.50 1.30 ± 0.15
Mean 59.95 ab
I. 66.20 40.25 – 108.85 1.28 ± 0.13
4 II. 30.15 19.70 – 46.15 1.78 ± 0.13
III. 35.60 26.20 – 48.45 2.19 ± 0.10
Mean 43.98 ab
I. 118.95 76.65 – 184.60 1.58 ± 0.12
5 II. 241.80 103.00 – 567.65 0.84 ± 0.17
III. 279.50 120.15 – 650.10 1.13 ± 0.20
Mean 213.42 cd
I. 84.95 55.30 – 130.50 1.38 ± 0.11
6 II. 74.30 50.00 – 110.40 1.59 ± 0.11
III. 181.00 136.75 – 239.60 2.12 ± 0.09
Mean 113.42 abc
I. 14.60 8.35 – 25.20 0.99 ± 0.17
7 II. 24.40 17.45 – 34.10 1.42 ± 0.12
III. 51.80 34.60 – 77.55 1.49 ± 0.11
Mean 30.27 ab
I. 214.25 157.50 – 291.45 1.91 ± 0.10
8 II. 175.25 127.25 – 241.50 1.93 ± 0.10
III. 253.10 179.20 – 357.55 1.70 ± 0.10
113
Table 7 continues
Mean 214.20 cd
I. 56.20 32.90 – 95.95 1.17 ± 0.13
9 II. 30.00 19.65 – 45.85 1.47 ± 0.11
III. 19.60 9.90 – 38.75 1.05 ± 0.15
Mean 35.27 ab
I. 103.15 72.50 – 146.80 1.80 ± 0.11
13 II. 213.45 103.95 – 438.35 1.11 ± 0.17
III. 76.65 44.65 – 131.55 1.19 ± 0.13
Mean 131.08 bcd
I. 212.70 164.85 – 274.40 1.80 ± 0.11
14 II. 89.30 57.65 – 138.30 1.38 ± 0.12
III. 216.10 138.75 – 336.55 1.46 ± 0.12
Mean 172.70 cd
I. 339.40 128.15 – 899.00 1.39 ± 0.25
16 II. 165.55 88.4 – 309.95 1.22 ± 0.15
III. 192.80 155.05 – 239.75 2.00 ± 0.10
Mean 232.60 d
I. 126.50 89.15 – 179.55 1.89 ± 0.11
17 II. 241.45 102.65 – 568.10 1.00 ± 0.19
III. 214.55 118.00 – 390.10 1.37 ± 0.16
Mean 194.17 cd
I. 297.75 177.20 – 500.35 1.45 ± 0.14
18 II. 148.50 95.65 – 230.55 1.39 ± 0.11
III. 154.85 116.85 – 205.20 1.57 ± 0.11
Mean 200.37 cd
I. 158.25 112.85 – 221.95 1.77 ± 0.10
19 II. 207.30 154.35 – 278.35 2.01 ± 0.10
III. 256.00 182.65 – 358.80 1.72 ± 0.10
Mean 207.18 cd
I., II., III. – three replicates of the bioassay
* Means followed by the same letter are not significantly different (Tukey’s HSD test, P < 0.05)
** SE – standard error of the mean
5.2.3 GERMINATION OF PRIMARY CONIDIA

Results of conidial germination experiment are shown in the Table 8. Generally, primary
conidia of the isolates could germinate on all the surfaces and both the relative humidities.
However, a character of germination varied between the surface types and the relative
humidity regimes. As for the relative humidity it was evident that more of the conidia
germinated at saturated atmosphere. At this level of humidity conidia of all the isolates
germinated on each surface type. No germination was, however, recorded on nutrient agar
(YG) for isolates 7, 17 and 19. At 75% relative humidity conidia of majority of isolates did
not germinate on a glass surface, or only few conidia germinated. On the contrary, conidia
germinated well at 75% relative humidity on water agar (WA) and YG agar. For P.
neoaphidis a strong dependence of sporulation /WILDING 1969/ and germination /MORGAN et
al. 1995, SIVČEV and MANOJLOVIĆ 1995, SIEROTZKI et al. 2000/ on relative humidity near to
or at saturation was reported. High relative humidity is prerequisite for conidial germination.
P. neoaphidis conidia were reported to be able to germinate only at high relative humidity. At
93% of relative humidity there was no germination and the conidia germinated faster at 100%
than at 97% of relative humidity /SIVČEV and MANOJLOVIĆ 1995/. Our results confirmed the
necessity of saturation for conidial germination. We assume that the higher proportion of
germinating conidia recorded at 75% relative humidity on the agar substrates was probably
due to water present in the media, which might have compensated the low air humidity in the
vicinity of conidia. Since in the course of the germination assay on glass the alternative source
of water was missing the germination was absent or low. Airborne conidia are considered
114
very short-lived structures /HAJEK and ST. LEGER 1994/. For infection to start, after conidia
are produced and discharged, they must survive until contacting a new host. We observed that
conidia exposed to low humidity (75%) for 4 hours did not germinate. The germination did
not occur either the conidia were subsequently exposed to 100% relative humidity (not in the
Table 8). Correspondingly, UZIEL and KENNETH /1991/ found that primary conidia could not
tolerate prolonged exposure to low relative humidity. BROBYN et al. /1987/ demonstrated that
P. neoaphidis conidia could survive prolonged period of 40-50% relative humidity and 90%
relative humidity, while infectivity declined rapidly at 70% relative humidity. However, the
primary conidia remained infective for at least 14 days in field during summer /BROBYN et al.
1985/. Conidial viability of Z. radicans determined by methods of vital fluorochrome staining
did not change significantly after 4 hour exposure to 75% of relative humidity /GRIGGS et al.
1999/.
After landing on host or non-host surface, the primary conidium of P. neoaphidis produce
either a secondary conidium, a germ tube, or an appressorium /BUTT et al. 1990, UZIEL and
KENNETH 1999/. We evaluated the first two types of conidial germination on the non-host
surfaces. Appressorium formation was not quantified, since appressoria are more evident 12
hours after inoculation /BUTT et al. 1990/ and we made observation after 4-hour incubation
period. Moreover, appressoria are difficult to distinguish accurately at magnification we used
and they are best detected by fluorescence microscopy /BUTT et al. 1990/. P. neoaphidis
appressoria are significantly more produced on living substrates (aphid cuticle and leaves)
than on glass /DARA and SEMTNER 1998/. Our results indicate that character of surface can
influence a type of conidial germination. At 75% of relative humidity secondary conidia were
mostly produced and only few germ tubes were produced in isolates 9, 13, 16, 17, and 18.
However, a level of germ-tube formation did not exceed 9% and was 1-2% in most isolates.
At saturated atmosphere the germ-tube formation prevailed over the secondary conidium
formation on the glass surface, whereas secondary conidia were almost exclusively produced
on the agar surfaces. Recently, similar experiment has been carried out by SIEROTZKI et al.
/2000/. They also found out secondary conidium production on hydrophilic agar substrate and
germ-tubes were produced predominantly on hydrophobic polystyrene surface. They even
observed a formation of secondary conidia on glass slides when water condensated on the
slides and contacted the conidia. It was hypothesized that the presence or absence of free
water, rather than physico-chemical properties of the surface, is the trigger for differentiation
into secondary conidia or germ-tubes. However, in our experiments water condensed on glass
slides, but mostly germ-tubes were produced. We can agree that surface hydrophobicity is not
a cue for the differentiation of conidial germination, or at least, a contribution of surface
hydrophobicity to the germination type is questionable. HAJEK et al. /2002b/ also observed
that rather chemical stimuli and not changes in hydrophobicity of surface affected
germination of conidia of Entomophaga maimaiga Humber, Shimazu et Soper. Germination
of infective conidia on various surfaces has been studied for a number of entomopathogenic
fungi /e.g. STEINKRAUS and SLAYMAKER 1994, SIVČEV and MANOJLOVIĆ 1995, GRIGGS et al.
1999, UZIEL and KENNETH 1999, SIEROTZKI et al. 2000, KALSBEEK et al. 2001, HAJEK et al.
2002b, 2002a/ and influence of diverse physico-chemical and nutritional parameters on
stimulation and differentiation of conidial germination were evaluated. At optimal conditions
(temperature and humidity) the process of germination can be affected by nutrients or
chemical stimuli /SAMPEDRO et al. 1984, LATGÉ et al. 1987, BOUCIAS and LATGÉ 1988,
NADEAU et al. 1996a, GRIGGS et al. 1999, UZIEL and KENNETH 1999/, by conidial density on
the surface /BREY et al. 1986, HAJEK et al. 2002a/, or by rigidity of surface /HAJEK et al.
2002b/.
Entomophthoralean fungi infect their hosts principally by means of forcibly discharged
primary conidia via integument /BREY et al. 1986, BUTT et al. 1990, HAJEK and ST. LEGER
115
1994/. Under favourable environmental conditions the conidia land on host surface and
germinate to penetrate the integument and initiate the infection process /BREY et al. 1986,
BUTT et al. 1990/. Theoretically, a high vitality of conidia (a high conidial germination) of
certain fungal strain positively affects mortality of host insect. In order to evaluate the
relationship between conidial germination and virulence of the Slovak isolates a regression
analysis was performed. The Figure 23 shows a result of the linear regression between lethal
concentration and germination of P. neoaphidis conidia on glass surface at 100% relative
humidity. Correlation coefficient (-0.9356) indicates a strong negative relationship between
the fungus characteristics. The relationship was statistically very significant (t 3.67 =9.55,
P=0.001, df=28). However, there was only weak correlation detected for germination on
water agar, or YG agar at 100% relative humidity (-0.0088, or -0.3273, respectively).
Association between conidium germination and virulence was presented by
KHACHATOURIANS /1996/, as well. The strong correlation implies that the level of conidial
germination on glass could be comparable to that of germination of conidia in vivo on host
cuticle. Though, the in vivo germination may not be equal to that of in vitro because of
different environment and various factors involved. HAJEK et al. /2002a/ found that conidia of
E. maimaiga on the larval cuticle produced germ tubes very rapidly when compared with
conidia on water agar, suggesting that some stimulants were present. It is well known that
cuticular lipids of host influence conidial germination of C. obscurus /SAMPEDRO et al. 1984,
BOUCIAS and LATGÉ 1988/ or Erynia conica /NADEAU et al. 1996a/. HOUNTONDJI et al. /2002/
also demonstrated that capilliconidia of Neozygites floridana (Weiser et Muma) did not
germinate on non-host cuticle. Presumably, not all conidia, which have landed on host cuticle,
penetrate the integument. Number of penetrating germ tubes of C. obscurus was lower than
10% of the germinating conidia on pea aphid cuticle and when the number of conidia on
cuticular surface exceeded 150-200 per aphid, or if conidia were clumped together, the level
of germination was extremely low producing only a few secondary conidia /BREY et al. 1986/.
We assume that the strong correlation presented here rather gives evidence about the conidial
vitality expressed by high level of germination on non-host surface.
Figure 23. Linear relationship between lethal concentration LC 50 (conidia/mm2) and germination of
conidia (%) of P. neoaphidis isolates on glass surface
250
200
LC 50 (conidia x mm )
-2
y = -2.1659x + 233.51
y = -2,1659x + 233,51
150
100
50
0
0 20 40 60 80 100 120
Conidial germination on glass (% )
116
Table 8. Germination of P. neoaphidis primary spores on two surfaces and at two humidity regimes
Isolate Germination 100% relative humidity 75% relative humidity

number type Glass WA YG Glass WA YG
Germ tube 76% 0% 0% 0% 0% 0%
1 Secondary conidium 23% 93% 85% 0% 100% 96%
∑ 99% 93% 85% 0% 100% 96%
Germ tube 3% 1% 0% 0% 0% 0%
∑ 4% 97% 10% 0% 90% 0%
Germ tube 50% 0% 0% 0% 0% 0%
∑ 50% 14% 27% 0% 83% 15%
Germ tube 83% 0% 0% 0% 0% 0%
∑ 83 91% 84% 0% 97% 91%
Germ tube 4% 1% 0% 0% 0% 0%
∑ 4% 44% 63% 0% 90% 51%
Germ tube 54% 0% 1% 0% 0% 0%
∑ 54% 96% 1% 0% 100% 0%
Germ tube 19% - 0% 0% 0% -
7 Secondary conidium 75% - 0% 0% 100% -
∑ 94% - 0% 0% 100% -
Germ tube 28% 0% 0% 0% 0% 0%
∑ 30% 81% 61% 7% 89% 0%
Germ tube 35% 0% 0% 1% 0% 0%
∑ 83% 95% 98% 1% 96% 92%
Germ tube 5% 8% 0% 0% 2% 0%
∑ 65% 71% 34% 0% 83% 58%
Germ tube 10% 0% 0% 0% 0% 0%
∑ 26% 26% 10% 0% 58% 41%
Germ tube 8% 5% 0% 1% 0% 0%
∑ 15% 79% 59% 1% 92% 85%
Germ tube 42% 0% 0% 2% 2% 0%
∑ 42% 96% 0% 2% 94% 0%
Germ tube 19% 0% 0% 9% 0% 0%
∑ 19% 88% 1% 9% 87% 4%
Germ tube 21% 0% 0% 0% 0% 0%
∑ 21% 89% 0% 0% 90% 1%
Glass – cover glass; WA – water agar (2%); YG - 2% yeast extract, 3% glucose, 1.5% agar; - data not available
5.2.4 RADIAL GROWTH RATE

P. neoaphidis isolates showed a good growth on the culture medium. Daily rate of the radial
growth ranged from 35.03 to 116.67 mm2. A one-way ANOVA showed significant difference
in radial growth rate among the isolates tested at 99% confidence level (F 2.39 = 22.11, P >
117
0.01). Multiple range tests showed heterogeneity in the results (Table 9), and 9 homogenous
groups were detected. Isolates of the same origin have also significantly different growth rate.
MILNER et al. /1983/ stated that growth rates could be difficult to evaluate accurately since the
results varied unpredictably between experiments. Pattern of radial growth dynamics was
similar for all Slovak isolates. The radial growth increased till 25th or 30th day of the
experiment. After the peak the growth rate decreased until the plate was completely covered
by fungus colony, or until the end of experiments on the 35th day.
Table 9. Radial growth rate of the Pandora neoaphidis isolates
Isolate Mean radial growth increase (mm2) / days after a plate inoculation
Daily rate
th th th th th th th
number 5 day 10 day 15 day 20 day 25 day 30 day 35 day of growth
1 158.23 301.00 478.86 629.36 743.95 518.20 - 94.32 gh*
2 19.71 85.51 152.21 242.85 254.82 516.49 - 42.39 ab
3 31.68 41.05 148.79 489.12 472.02 357.44 444.66 56.71 bc
4 33.39 90.64 206.94 244.56 699.48 595.16 636.20 71.61 def
5 26.55 85.51 111.16 237.72 184.70 405.32 - 35.03 a
6 105.22 295.87 417.29 579.77 632.78 848.27 - 95.97 h
7 26.55 88.93 359.15 480.57 593.45 547.27 492.54 73.96 def
8 112.06 126.56 210.36 381.38 613.97 796.96 721.71 84.66 fgh
9 59.04 152.21 401.90 656.73 831.17 814.07 415.58 95.16 gh
13 100.09 258.24 417.29 338.62 578.06 641.33 482.28 80.45 efg
14 67.59 95.77 177.86 141.95 242.85 331.78 285.61 38.38 a
16 84.69 182.99 294.16 523.33 670.41 527.32 - 69.07 cde
17 65.88 75.25 222.33 208.65 335.20 321.52 - 40.96 a
18 315.57 386.51 708.03 588.32 918.39 - - 116.67 i
19 36.81 63.28 225.75 360.86 465.18 538.72 425.85 60.47 cd
* Means within column followed by the same letter are not significantly different (Tukey’s HSD test, P<0.05)
5.2.5 BIOMASS PRODUCTION OF THE ISOLATES

The P. neoaphidis isolates could be easily Table 10. Average biomass production
cultivated in liquid media. Laboratory-scale of P. neoaphidis isolates
bioassay was carried out to evaluate and
Isolate Average biomass
quantitatively compare a mycelial production of the
number production (g.l-1)
isolates. Results of the experiment are shown in the
Table 10. Average biomass production of P. 1 8,40 d*
neoaphidis isolates ranged between 1.63 to 8.72 g.l- 2 3,42 b
1
. The biomass production was statistically different 3 3,78 b
among the isolates (F 2.32 = 29.86, P > 0.01). 4 3,95 b
Tukey’s HSD test identified heterogeneity in the 5 1,82 a
results and 4 homogenous groups were determined. 6 8,32 d
It is of interest that isolates of the same origin 7 5,80 c
(isolates of numbers 6,7 and 8,9) produce 8 1,83 a
significantly different amount of biomass at 9 5,32 c
standard conditions. SIEROTZKI et al. /2000/ 13 7,93 d
published the biomass production of Swiss material 14 3,23 b
118
in the range of 7.8-14.9 g.l-1. Even though 16 3,25 b

we used liquid medium of the same 17 1,63 a
composition in the experiment, the Slovak 18 8,72 d
isolates yielded fewer biomass. The Swiss 19 6,57 c
isolates also showed significant differences
in biomass production irrespective of isolate *Means followed by the same letter are not
significantly different (Tukey’s HSD test, P < 0.05)
origin /SIEROTZKI et al. 2000/. We found a
strong positive correlation (+0.737; t 3.67 =3.93, P=0.001, df=28) between the average biomass
production and the daily rate of radial growth (Figure 24).
Figure 24. Linear regression between biomass production (g.l-1) and radial growth (mm2.day-1) of P.
neoaphidis isolates
12
Mean biomass pr oduction (g.l-1)
10
6 y = 0,0761x
y = 0.0761x - 0,4253
– 0.4253
0
0 20 40 60 80 100 120 140
Daily rate of radial growth (mm 2)
5.2.6 VARIABILITY OF PANDORA NEOAPHIDIS ISOLATES

Selection of a virulent isolate for augmentation was not the objective of this study, but
rather an assessment of isolate variability. We evaluated phenotypic characters of P.
neoaphidis isolates and the results revealed a significant intraspecific variation within this
species. Intraspecific variability of entomophthoralean fungi was often characterised by
differences in pathogenicity /PAPIEROK 1982/. Recently, molecular techniques has been
involved to analyse intraspecific variation in the fungal isolates /ROHEL et al. 1997, SIEROTZKI
et al. 2000/. The Slovak isolates varied significantly in all the phenotypic characters
evaluated. Even, isolates obtained from single colonies during epizootics shoved significant
differences in the properties investigated, except that of conidial size. This may indicate that
epizootics in the field are caused by consortia of strains and not by prevalence of a single
virulent strain. Within-field variability of P. neoaphidis strains has been recently discussed by
SHAH et al. /2004/. Results of molecular analyses by ROHEL et al. /1997/ and SIEROTZKI et al.
/2000/, who found variability not only in strains of wide geographic origin, but also between
isolates originating from the same field and host, support the association of more fungus
strains during epizootics.
119
5. RESULTS AND DISCUSSION: 5.3 Aphid-pathogenic Entomophthorales at Dolná Malanta
5.3 APHID-PATHOGENIC ENTOMOPHTHORALES AT DOLNÁ MALANTA

5.3.1 PREVALENCE OF APHID-PATHOGENIC ENTOMOPHTHORALES AT DOLNÁ MALANTA
Besides the extensive surveys of aphid pathogenic Entomophthorales throughout the
country, a regular monitoring of aphid species and their pathogens was carried out at the
locality of Dolná Malanta over two years. The study included a variety of habitat types
(cultivated and non-cultivated vegetations) with, altogether, 18 aphid species regularly
sampled on 19 host plants (Table 2, see section 4.4.2). Each habitat type possesses specific
properties to support both aphids and their fungal enemies.
Acyrthosiphon pisum: Two fungal pathogens from the order Entomophthorales were
found in the aphid populations on field pea. They were identified as P. neoaphidis and C.
obscurus. While two P. neoaphidis and three C. obscurus-killed aphids were only found in
the 2001 sampling season, an epizootic development of mycoses was detected in 2002 (Table
56, see appendix). In 2002 P. neoaphidis was the predominant species with up to 40%
mortality recorded. These results are consistent with those published by CAGÁŇ and BARTA
/2001/ who found out the same fungal pathogens from the pea aphid at this locality. The two
fungal species are reported all over the world as more or less regular reducing factors of pea
aphid populations, but levels of infection may vary among years, localities, and populations.
In Slovakia P. neoaphidis was recorded from pea aphid for the first time in the course of the
aphid outbreak on alfalfa during the twenties of the last century /VIELWERTH 1921 in STARÝ
1974, DRASTICH and ROZSYPAL 1927 in STARÝ 1974/. C. obscurus and P. neoaphidis were
recorded from A. pisum in the Great Britain, Poland, Russia, the USA, Canada, and Australia
/e.g. MACLEOD 1955, VORONINA 1971, BROBYN and WILDING 1977, MILNER 1982,
PICKERING et al. 1989, BALAZY et al. 1990, PICKERING and GUTIERREZ 1991/. Many of these
authors found epizootics within pea aphid populations so that pathogens are considered as
important biological agents for aphid control /WILDING 1975, PICKERING et al. 1989, KISH et
al. 1994/. In the world there are further three entomophthoralean species reported from the
pea aphid. They are E. planchoniana, N. fresenii, and Z. radicans. Generally, they are
considered minor pathogens of the pea aphid /KENNETH and OLMERT 1975, WILDING 1975/.
CAGÁŇ and BARTA /2001/ observed P. neoaphidis activity among pea aphid colonies in the
alfalfa crops, as well. Though, the number of fungus-killed aphids was apparently low in one
year and no infected aphids were found in the following one. The alfalfa and pea plots in our
experiments grew nearby, so we expected high infestation of alfalfa by aphids and a share of
natural enemies between the colonies. During our routine sampling, however, a very low
infestation of alfalfa crop by the aphid (infestation level < 0.5 aphids per SU) was observed in
both years (data not presented) and no fungus-killed aphids were identified. The low
infestation may be due to lower nutritional attractiveness of alfalfa in comparison with the
field pea. We assume that the low incidence of host may be the reason for zero infection.
CAGÁŇ and BARTA /2001/ recorded entomophthoralean infection in alfalfa crop though the
infestation level did not exceeded 2 aphids per SU.
Aphis fabae: Populations of the black bean aphid and their diseases were evaluated on the
primary host, Euonymus europaea, and three secondary hosts, Faba vulgaris, Beta vulgaris,
and Arctium lappa growing at the locality. We found annual variation in the host infestation,
aphid infection rate, and species diversity of entomopathogenic fungi attacking the black bean
aphid populations (Tables 57, 65 and 66, see appendix). Altogether four entomophthoralean
species were identified from the aphid at the locality; N. fresenii, E. planchoniana, P.
neoaphidis, and C. obscurus. Up to now seven fungal species from the order
Entomophthorales took part in the natural control of A. fabae in various countries. They are N.
fresenii, P. neoaphidis, E. planchoniana, C. obscurus, C. destruens, C. coronatus, and E.
nouryi /e.g. BATKO 1962, GUSTAFSSON 1969, THOIZON 1970, KREJZOVÁ 1972, KENNETH and
120
OLMERT 1975, BEN-ZE’EV and KENNETH 1979, KELLER and SUTER 1980, BAŁAZY 1993,
STEENBERG and EILENBERG 1995/. Several attempts have also been made to introduce aphid-
pathogenic Entomophthorales into laboratory or field populations of the black bean aphid /e.g.
KREJZOVÁ 1972, DEDRYVER 1979, WILDING et al. 1986/. Under conditions of Slovakia a
record by WEISMANN et al. /1961/ on a positive role of Entomophthorales in reduction of the
black bean aphid population is the only reference to these organism in A. fabae colonies.
In spite of a relatively high infestation of the primary host by aphids, entomophthoralean
infection was negligible within these colonies in both years. The rate of infection of black
bean aphid on the perennial host was weak in springs and autumns, although a species
spectrum was greater in autumns. In spring, one alate aphid was merely found being killed by
N. fresenii in 2001 and similarly one alate aphid was killed by E. planchoniana in 2002. In
autumn, mortality of aphids due to the diseases was also weak, but in contrast, three
entomophthoralean fungi were identified; N. fresenii, E. planchoniana, and P. neoaphidis.
Similarly, KISH et al. /1994/ observed a low mortality of Myzus persicae Sulzer on peach trees
(a primary host) despite high densities of aphids on trees in spring and autumn. However, a
considerable suppression of green peach aphid populations was occurred on potatoes and non-
solanaceous hosts by fungi in summer. Other authors likewise referred a low infection of
fundatrices, as well. Only three apterous adults of A. fabae on a winter host were infected by
E. planchoniana in spring /WILDING and PERRY 1980/. The authors found also only few
individuals killed by the pathogen during the autumn.
One pathogen, N. fresenii, was identified in aphid colonies on field bean in 2001. In the
following year besides N. fresenii two other species, P. neoaphidis and C. obscurus, were
identified, as well. Out of the pathogens observed within the aphid colonies N. fresenii was
the most effective fungus. Aphids on sugar beet were killed by the same composition of three
entomopathogens. N. fresenii and C. obscurus were recorded in 2001, whereas N. fresenii and
P. neoaphidis infected the black bean aphid in 2002. Mycoses occurred only in a low number
of aphids in the 2001 sampling season. On the contrary, epizootic development of N. fresenii
was recorded in 2002. N. fresenii appeared to be the dominant species on the black bean aphid
colonising plants of A. lappa in 2001. In these colonies P. neoaphidis was identified from one
aphid cadaver and no other fungal species were recorded.
In Slovakia, N. fresenii was the predominant species infecting the black bean aphid on all
the host plants and it appears to be the most important natural regulating factor of A. fabae.
The pathogen was reported as important mortality factor of the black bean aphid in Sweden,
as well /GUSTAFSSON 1969/. In western France, populations of A. fabae on field bean were
regularly destroyed by P. neoaphidis and N. fresenii /MISSONNIER et al. 1970/. According to
DEDRYVER /1978/ N. fresenii was the main pathogen of A. fabae in the west of France
whereas other species (P. neoaphidis, C. obscurus, and E. planchoniana) affected the aphids
on a small scale. In Poland P. neoaphidis was predominant among other species (C. obscurus,
C. thromboides, E. planchoniana, N. fresenii, and C. coronatus) infecting the aphid
/MAJCHROWICZ 1978/. In the same way, P. neoaphidis was responsible for epizootics in A.
fabae populations in field bean in the Netherlands /MIETKIEVSKY and VAN DER GEEST 1985/.
Conversely, in the Great Britain the most part of the aphid mortality was due to E.
planchoniana, though many aphids were killed by P. neoaphidis and C. obscurus, as well. N.
fresenii and C. thromboides killed very few aphids /WILDING and PERRY 1980/. Apparently,
there are three entomophthoralean species with epizootic potential in the black bean aphid
populations in Europe, N. fresenii, P. neoaphidis, and E. planchoniana. It is of interest that in
different countries different pathogens took part in disease outbreaks of the aphid populations.
In Slovakia, likewise in France or Sweden, it was N. fresenii that showed its potential for
epizootic development in the black bean aphid populations.
121
Cereal aphids: Entomophthoralean diseases of cereal aphids were investigated in aphid

colonies on primary hosts, Rosa canina (p. host for M. dirhodum) and Padus avium (p. host
for R. padi), and on the secondary hosts, Zea mays, Hordeum vulgare, and Triticum aestivum.
Five aphid species were recorded from the cereals or the winter hosts, R. padi, M. dirhodum,
S. avenae, D. noxia, and R. maidis. Abundance of aphids varied with year and host plant and
prevalence of Entomophthorales varied with year, host plant and host aphid (Tables 58, 60-
63,72, and 73, see appendix). Aphid species R. padi, M. dirhodum, and S. avenae are common
pests of cereals in Slovakia /e.g. CAGÁŇ 1991, ŠTALMACHOVÁ and CAGÁŇ 2000/. Recently,
however, new species of cereal aphids from Slovakia have been found. It was D. noxia on
summer barley /LUKÁŠ et al. 2001, TÓTH 2002/ and R. maidis on emerged remainders of
winter wheat in early autumn /CAGÁŇ and ŠTALMACHOVÁ 2001/. We recorded both these
aphid species at Dolná Malanta. D. noxia was infesting summer barley during both years and
whereas few individuals of R. maidis were observed on winter wheat in 2002, plenty of R.
maidis infested summer barley in the same year. Cereal aphids are frequently attacked by
entomophthoralean fungi /e.g. DEAN and WILDING 1971, 1973, DEDRYVER 1983, FENG et al.
1990, HATTING et al. 1999a/. Up to present three fungal species, P. neoaphidis, C. obscurus,
and E. planchoniana, were identified from cereal aphids in Slovakia /ŠTALMACHOVÁ and
CAGÁŇ 2000/. During this survey all the three species were observed and two additional
entomophthoralean fungi, N. fresenii and Z. aphidis, were identified from aphid cadavers at
the study area. Four main species of Entomophthorales, P. neoaphidis, C. obscurus, E.
planchoniana, and N. fresenii, are regularly recorded infecting cereal aphids in Europe
/COREMANS-PELSENEER et al. 1983, ŠTALMACHOVÁ and CAGÁŇ 2000/, in North America
/FENG et al. 1990, 1991/, or in North and South Africa /HATTING 1999a, 2000, ABDEL-
MALLEK et al. 2003, ABDEL-RAHMAN and ALI 2003/. C. coronatus, C. thromboides, E.
chromaphidis, Z. radicans, and Z. occidentalis are minor parasitic fungi of cereal aphids
/FENG et al. 1990, STEENBERG and EILENBERG 1995, HATTING et al. 1999a,b, 2000, ABDEL-
MALLEK et al. 2003/.
Abundance of R. padi on the primary host was spectacular in both years, although in
springs the abundance was commonly lower than in autumns. Few aphid cadavers were
observed in the colonies of fundatrices during springs and all of them were killed by E.
planchoniana. Conversely, colonies of R. padi were significantly attacked by a great diversity
of entomophthoralean fungi during autumns. In 2001 no individuals of M. dirhodum were
observed on its winter host, Rosa canina, during the spring, however the aphids appeared in
the autumn. In the 2002 spring fundatrices were infesting the shrubs and disappeared by
beginning of May. In the autumn the populations re-appeared. Fungus-killed aphids were only
present in the autumn generations, though in low numbers. P. neoaphidis and E.
planchoniana were identified from cadavers. All of randomly examined aphid cadavers were
infected by E. planchoniana in 2001 and by P. neoaphidis in 2002. Temporal distribution of
infection on the primary hosts for cereal aphids corresponds to that of infection on A. fabae, a
fungal prevalence is subtle in springs and increases in autumns.
Three cereal aphids were infesting the maize crop. They were R. padi, M. dirhodum, and S.
avenae. In 2001 R. padi dominated followed by M. dirhodum, and S. avenae. In contrast, M.
dirhodum was dominating in 2002, though R. padi was present in high numbers, as well.
Three fungi, P. neoaphidis, E. planchoniana, and N. fresenii, were infecting the cereal aphids
on maize. Infection rate was very low in 2001, but epizootic caused by P. neoaphidis was
recorded within colonies of M. dirhodum in 2002. During this year M. dirhodum clearly
exhibited high susceptibility toward P. neoaphidis. It is of interest that R. padi, the second
most abundant aphid, was virtually free of fungal infection (there was just one individual
killed by C. obscurus). This could be attributed to the fact that most of R. padi individuals
were found feeding on or under of maize cob bracts, thus minimizing the chances of coming
122
into contact with fungal inoculum (from sporulating M. dirhodum cadavers) present on maize
leaves. However, a part of R. padi population occupied the maize leaves, the same feeding
niche as M. dirhodum, thus being exposed to the same microclimatic conditions supporting
disease in M. dirhodum. Despite this no R. padi infection by P. neoaphidis was observed. This
might suggest some level of non-susceptibility in R. padi to the strain/strains of P. neoaphidis.
Similar lower susceptibility of R. padi to P. neoaphidis has already been observed by others
/DEDRYVER 1981, 1983, HATTING et al. 2000, SHAH et al. 2004/.
At the locality the winter wheat crop was infested by four aphid species, R. padi, R. maidis,
M. dirhodum, and S. avenae. R. maidis was not observed in 2001 and only 2 apteral
individuals were recorded in 2002. R. padi and S. avenae were recorded in substantial
numbers from the crop in 2001. On the contrary M. dirhodum and S. avenae were dominating
in 2002. P. neoaphidis, E. planchoniana, N. fresenii, and C. obscurus reduced the aphid
colonies on the winter wheat, but the infection rate was low. No cadavers of R. maidis were
found and likewise in the maize crop the colonies of R. padi were practically free of infection
except few cadavers detected in autumn population in 2001.
While D. noxia was the only cereal aphid recorded from the summer barley crop in 2001,
five cereal aphids, D. noxia, R. padi, R. maidis, M. dirhodum, and S. avenae infested the crop
during the following sampling season. Despite D. noxia is relatively new species for Slovakia,
it was recorded in extremely high abundance on the host exceeding infestation level of 80
individuals per SU in 2001. In the next year the species was not such abundant; at a peak of
population development the infestation reached about 2.5 aphids per SU. In 2002 M.
dirhodum and R. padi dominated followed by R. maidis, D. noxia, and S. avenae. Complex of
entomophthoralean fungus species has the same composition as that on aphids from winter
wheat with P. neoaphidis as prevalent fungus. The fungus was active especially within
colonies of D. noxia (in 2001) and M. dirhodum (in 2002).
The observations indicate a prime role of P. neoaphidis as a regulating factor of cereal
aphids in the locality with ability to epizootic regulation of hosts. This is in agreement with
results of other authors. DEDRYVER /1983/ found P. neoaphidis to be more efficient in
regulation of cereal aphids in France than C. obscurus or E. planchoniana. In Denmark P.
neoaphidis was the most common fungus among cereal aphids and it appeared earliest and
latest within host colonies /STEENBERG and EILENBERG 1995/. The species was also the most
prevalent in colonies of M. dirhodum and D. noxia in South Africa /HATTING et al. 2000/ and
the USA /FENG et al. 1990/. FENG et al. /1991, 1992a/ found P. neoaphidis epizootics more
frequently in the populations of M. dirhodum than those of S. avenae, which were more often
subjected to C. obscurus. They attributed these observations to different humid conditions of
microhabitats occupied by the aphid species. S. avenae prefers the upper leaves and ears
where apparently lower humidity might limit the development of P. neoaphidis infection.
However, there is a different observation by DEDRYVER /1981/ who found C. obscurus being
more pathogenic for aphids on leaves than for aphids on ears. We found S. avenae infected by
P. neoaphidis and E. planchoniana, though the fungal prevalence was low. No indication of
fungal preference based on aphid species was observed but that of M. dirhodum preference to
R. padi observed for P. neoaphidis.
Microlophium carnosum: Colonies of the nettle aphid were surveyed on patches of the
stinging nettle growing along an irrigation channel. Abundance of aphids varied among years
reaching the highest numbers in 1998 and 2001 (Tables 67-70, see appendix). P. neoaphidis,
N. fresenii, N. microlophii E. planchoniana, and C. obscurus were identified from the aphid.
The first three species were predominant with epizootic potential. Up to now 7
entomophthoralean fungi were reported from the aphid, P. neoaphidis, E. planchoniana, C.
obscurus, N. fresenii, N. microlophii, C. coronatus, and Z. phalloides /BATKO 1966a,
LEATHERDALE 1970, THOIZON 1970, MIĘTKIEVSKY and VAN DER GEEST 1985, KELLER
123
1987a, 1991, BAŁAZY et al. 1990, BAŁAZY 1993/. Seasonal prevalence and species variation
of the pathogens is discussed in more detail in the section 5.3.3 with intention to evaluate the
nettle patches as reservoirs for the pathogens in the landscape.
Myzus cerasi: Spring and autumn generations of the black cherry aphid were evaluated on
its primary host Cerasus avium. In both years aphid density in spring populations was more
abundant compared to the autumn populations (Table 59, see appendix). Few aphids were
found suffering from entomophthoralean infection. In 2001 three cadavers were recorded in
May and one at the end of October. In the following year no infection occurred in spring
generations and 13 cadavers were recorded in October. Altogether four pathogenic fungi were
identified, P. neoaphidis, N. fresenii, E. planchoniana, and Z. aphidis. It is of interest that
despite such high infestation of trees in springs, especially in 2001, mortality due to
entomophthoralean disease was imperceptible. Though, a rather high mortality was provided
by parasitism of hymenopteran parasitoids and mostly by predation of ladybirds and
hoverflies. Fungal parasitism of the black cherry aphid has been poorly studied. Preliminary
study on natural enemies of M. cerasi in Norway revealed three entomophthoralean fungi, P.
neoaphidis, E. planchoniana, and C. obscurus /KLINGEN and JAASTAD, 2003/. THOIZON
/1970/ reported E. conica from M. cerasi, though this fungus is known as pathogen on
members from the families Culicidae and Chironomidae /KELLER 1991, BAŁAZY 1993/.
Hyalopterus pruni: Populations of the mealy plum aphid were studied on the primary host,
Prunus domestica, and the secondary host, Phragmites australis (Tables 54 and 55, see
appendix). Extremely high infestation of the plum trees was observed during the spring in
both years. The colonising aphids were in many cases completely covering underside of plum
leaves. The aphids were annually encountered on the secondary host in considerable numbers,
as well. Autumn populations of aphids were not established on the trees. Though the
infestation was so significant, individuals suffering from the mycoses were rare. In the course
of two years four fungus-killed aphids were recorded. One aphid killed by N. fresenii was
found on plum tree at the end of May in 2001. Remaining three cadavers with E.
planchoniana infection were recorded from the reed in June. Mycoses from H. pruni are only
seldom reported in the literature. For instance, some N. fresenii killed individuals were
collected in South Africa and Israel /KENNETH and OLMERT 1975, HATTING et al. 1999a/ or E.
planchoniana cadavers were collected in France /THOIZON 1970/. Individuals of H. pruni
were artificially inoculated with C. obscurus, C. thromboides, and Basidiobolus sp. in Latvia
/ČUDARE 1988/. Uncommon infections of the insect may indicate a certain degree of
resistance to the mycoses originating most probably from the wax powder covering the whole
body of aphid.
Apple aphids: Two aphid species colonising apple trees were recorded, Aphis pomi and
Dysaphis plantaginea. Infestation of the apple trees were observed during both years and
colonising of the trees was obviously heavier in 2001 than in 2002 (Tables 49 and 50, see
appendix). In 2001 the aphids were more abundant during spring with only few aphids
recorded in the autumn. In the following year the aphids were mostly present in the autumn,
although in low numbers. A high diversity of fungal species identified from the aphids did not
reflect the low prevalence of fungus-killed aphids in the colonies. We identified E.
planchoniana from D. plantaginea and E. planchoniana, P. neoaphidis, N. fresenii, C.
obscurus, and Z. aphidis from A. pomi. E. planchoniana was recorded within spring colonies
and the other species in the autumn generations. So far only N. fresenii and E. planchoniana
were reported from France as pathogens of A. pomi and E. planchoniana as pathogen of D.
plantaginea /THOIZON 1970/. In laboratory conditions P. neoaphidis infected A. pomi
/MACLEOD 1955/.
Aphids on Rosa canina: Three aphid species were annually colonising shrubs of Rosa
canina (Tables 52 and 53, see appendix). They were Macrosiphum rosae, Metopolophium
124
dirhodum, and Chaetosiphon tetrarhodum. The first two aphid species were observed during
springs and autumns, but M. rosae was usually more abundant. Ch. tetrarhodum was
observed during summer months, from half of June till beginning of October. In the course of
survey two fungal species were infecting the aphids, E. planchoniana and P. neoaphidis. No
mycosed M. rosae were observed in 2001 and in the following year one M. rosae was killed
by E. planchoniana. Colonies of M. dirhodum were attacked by Entomophthorales in the
autumns; E. planchoniana was identified in 2001 and P. neoaphidis in 2002. While no
infected Ch. tetrarhodum were found in 2002, several individuals succumbed to E.
planchoniana disease in 2001. This is probably the first report of entomophthoralean infection
from this aphid species. M. rosae is often found infected by E. planchoniana or P. neoaphidis
/LEATHERDALE 1970, THOIZON 1970, BAŁAZY et al. 1990, STEENBERG and EILENBERG 1995,
NIELSEN et al. 2001b/ and P. neoaphidis epizootics can be observed as well /BAŁAZY et al.
1990/. The cereal aphid, M. dirhodum, is also often a host for Entomophthorales /e.g. DEAN
and WILDING 1971, 1973, FENG et al. 1990/.
Anoecia corni: Colonies of A. corni were monitored on the aphid’s primary host, Cornus
sanguinea (Table 71, see appendix). Generally, populations of fundatrices were not as
abundant as populations of sexuales. Autumn generations of the aphids were infected annually
by one fungal species, Z. aphidis. A. corni is the type host for this fungus and colonies of the
aphids are often attacked by the fungus in Europe /REMAUDIÈRE and HENNEBERT 1980,
KELLER 1991, BAŁAZY 1993/.
Aphis sambuci: Populations of the aphid were studied on the primary host, Sambucus
nigra (Table 51, see appendix). The shrubs were highly infested by aggregated aphid colonies
during springs, especially in May, however only few individuals were killed by pathogens.
Altogether, we identified two fungal species, E. planchoniana and P. neoaphidis. The first
species infected more individuals. E. planchoniana was also reported from A. sambuci by
THOIZON /1971/.
Periphyllus testudinaceus: P. testudinaceus infested their host, Acer campestre, from the
end of March till the end of May and during October (Table 64, see appendix). Each year only
one infected aphid was recorded in the autumn. In 2001 the cadaver was infected by P.
neoaphidis, and in 2002 N. fresenii was identified from the cadaver.
Cavariella pastinacae: Colonies of the aphids were observed on twigs of Salix sp. in 2001.
The aphids appeared at the beginning of April and the abundance of population peaked in
mid-May (Table 57, see appendix). At the end of May several aphids killed by E.
planchoniana were recorded. In the following year no aphids were observed on the trees. In
France the aphid were found infected by E. planchoniana and P. neoaphidis /THOIZON 1970/.
At the locality aphids as a whole used different habitat types during the seasons because
the suitability of the habitat types changed over the growing seasons. The seasonal aphid
population pattern for most species can be generalised to exhibit a typical growth curve with
two peaks, spring and autumn ones. At the locality the highest numbers of aphids were
present from mid-May to mid-June due mostly to outbreaks of populations on cereals, field
pea, and field bean. The autumn outbreaks were due to propagation of sexuales on winter
hosts of dioecious species. Sometimes spring generations on primary hosts may also multiply
excessively to create an additional peak. Abundance of cereal aphids and the black bean aphid
sampled at Dolná Malanta and prevalence of fungal pathogens are presented in the Figures 25
and 26. It is obvious that prevalence of entomophthoralean infection simply reflects the
patterns of live aphids, though the peaks are about a week delayed. This can be generalised
for all aphid-pathogen systems. Altogether, 6 parasitic fungi out of three families were
identified from aphid cadavers at the locality during the regular sampling. They were P.
neoaphidis, Z. aphidis, E. planchoniana (family Entomophthoraceae), N. fresenii, N.
microlophii (family Neozygitaceae), and C. obscurus (family Ancylistaceae). Moreover,
125
Figure 25. Number of live and fungus-killed cereal and black bean aphids (per 45 SU) sampled on
primary hosts and secondary hosts (maize and broad bean, respectively) at Dolná Malanta in 2001
Figure 26. Number of live and fungus-killed cereal and black bean aphids (per 45 SU) sampled on
primary hosts and secondary hosts (maize and sugar beet, respectively) at Dolná Malanta in 2002
further two fungus species were identified from aphids collected accidentally at the locality.
They were Z.radicans from Brevicoryne brassicae, and E. erinacea from Aphis umbrella.
Species compositions of fungal pathogens on aphids sampled at the locality are showed in the
Figures 27 and 28. By count of aphid species susceptible to the fungi and the number of
sampling occasions when the fungi were recorded within host populations the fungal species
could be arranged in this order: P. neoaphidis > N. fresenii > E. planchoniana > C. obscurus
> Z. aphidis > N. microlophii. Monitoring of the aphid fauna and its pathogens in the
environment also provides data on the natural host preferences and seasonal fluctuations of
the different fungal species. P. neoaphidis, the most frequent pathogen at the locality, was
identified from 12 aphid species, and epizootics in colonies of A. pisum and M. dirhodum
were observed. In 2001 P. neoaphidis was the first species identified among the aphid
colonies. During the springs this fungus kept its dominant position among other fungi,
however, during the autumns prevalence of all the fungal species was more equal. In 2002 the
fungal composition had a similar pattern, though P. neoaphidis appeared about one month
126
Figure 27. Species composition of fungal pathogens on aphids sampled at Dolná Malanta in 2001 (n =
a total number of fungus-killed aphids recorded at the locality for all SU evaluated)
149
331
422
102
157
n=
10
48
16
98
81
75
56
95
87
39
8
Figure 28. Species composition of fungal pathogens on aphids sampled at Dolná Malanta in 2002 (n =
a total number of fungus-killed aphids recorded at the locality for all SU evaluated)
4387
3862
1002
277
137
69
71
96
14
12
12
n=
2
127
later. Even if E. planchoniana infected, in general, more aphid species (11 species) than N.
fresenii (9 species), N. fresenii cadavers were more prevalent in aphid colonies. This probably
relates with tendency of N. fresenii to attack aphids in dense colonies, primarily those of A.
fabae, with epizootic manifestation resulting in a collapse of entire colonies. On the contrary
E. planchoniana cadavers were found mostly solo and the disease never spread around. No
epizootics were observed. Moreover, the species was more active in autumn generations on
winter hosts. Some authors say that N. fresenii, in contrast to P. neoaphidis, E. planchoniana,
or Z. phalloides, seems unable to persist in enzootic form in small aphid populations
/REMAUDIÈRE et al. 1981/ and is more host-dependent /WILDING and PERRY 1980, RABASSE
and DEDRYVER 1982/. Further it is usually reported as being a somewhat atypical
entomopathogenic fungus, since it causes epizootics during relatively dry and hot periods
/GUSTAFSSON 1969, THOIZON 1970, DEDRYVER 1978, STEINKRAUS et al. 1991/. At Dolná
Malanta, normally, N. fresenii infection appeared a few weeks later when compared with the
first entomophthoralean infection was recorded in the season. This probably indicates
requirements for higher temperature to initiate the fungal activity in the environment. C.
obscurus was identified from 7 aphid species and likewise E. planchoniana and the fungus
was present in host colonies in enzootic form. C. obscurus was annually observed in pea
aphid populations and sporadically was identified from other aphid species. Z. aphidis was
identified from four aphid species, although A. corni was the only aphid, which was regularly
attacked by the fungus.
Several aphid species of agricultural importance overwinter on woody plants distributed in
the landscape, e.g. A. fabae, R. padi, or M. dirhodum. Aphids on the alternative host plants
may therefore be important for both overwintering of the fungi and, during the growing
season, directly for epizootic development in the aphid populations in the crops. The
hypothesis, by which fundatrices might be a source of fungal inocula for summer aphid
generations on secondary hosts, was not vindicated in this study because of poor fungal
activity in colonies of fundatrices during the springs. On the other hand, it was impossible to
decide, which infections occurring in summer populations were true primary ones, i.e. which
came from the fungal material in the soil, and which had been transmitted to crops by infected
but still mobile migrantes alatae. KRÁĽOVIČ /1970/ said that the fungi did not threaten the
fundatrices of pea aphid and they might be observed usually at the end of the outbreak, when
the aphids were generally weakened by ecological conditions.
Based on the two years data it is difficult to measure a positive effect, which these fungi
pose to aphids in the agricultural landscape. As a rule, the infections occurred relatively late,
in high population densities, when the economic thresholds had been exceeded. At the locality
the pathogenic fungi attacked the aphids only after they had become damaging on the crops.
This phenomenon was also reported in aphid colonies by other authors /e.g. GUSTAFSSON
1969, SIVČEV 1981, PELL et al. 2001/. For instance, the action threshold, the aphid incidence
level when pesticide application is recommended, for field bean and sugar beet is when 5% of
plants are infested with aphids. In 2001, the threshold for field bean and sugar beet was
exceeded already on the first sampling date, but the infection appeared as late as infestation
rate reached 80% (field bean plot) or 40% (sugar beet plot). In 2002, the situation was very
similar and the aphid infection increased after the peak of aphid abundance in the crops. The
action threshold for field pea is when, on average, 3-5 aphids are recorded per plant. In 2001
the level was not exceeded at all and in the following year the threshold was surpassed on 21
May. However, the pathogens started to be effective one month later. In 2001 abundance of
leaf aphids on summer burley reached a level for chemical control (> 25 aphids per tiller) on
30 June, but entomophthoralean infection was weak until mid-July. Whether this delay
represents a failure of the natural mortality factor to respond to the population build up of
hosts is not certain. The number of aphids required in order to obtain infection does not
128
appear to be very large /SHANDS et al. 1963, PICKERING and GUTIERREZ 1991/. It may simply
represent failure of all the necessary conditions for epizootic development to be present. It is
clear that a low population density of aphids is only required for an initial infection if the
other circumstances are favourable, i.e. properties innate to aphids, weather conditions, or
density of fungal inocula. Usually, the population densities were decreasing when the rate of
aphid infection began increasing and it was not possible to determine how much the disease
contributed to this decline. We can agree with SHANDS et al. /1963/ who state that the
establishment of the fungi in aphid colonies take a long time and that the influence on the
insect populations afterwards is rapidly noticed. Our results indicate that the pathogens do not
play important role in reduction of crop damage from aphids and their presence in the
colonies is most probably a consequence of strong propagation of pest. It is possible to
conclude that an outbreak of pathogens among aphid colonies just indicates a reduction of
aphid population after its peak and do not bring about its extinction. It can be concluded that
simply rely on natural outbreak of entomophthoralean diseases in aphid colonies as
mentioned, for instance, in the southern USA /STEINKRAUS et al. 1995, 1996a, 1998a/ cannot
be applied in Slovakia and rather some programmes of biocontrol should be implement within
IPM.
5.3.2 EPIZOOTIC PATTERNS OF ENTOMOPHTHORALEAN DISEASES AND SPATIAL DISTRIBUTION OF

APHIDS
In the area of Dolná Malanta the intensive monitoring of variety of aphid species and their
fungal parasites (see section 5.3.1) revealed epizootic disease development in some host-
pathogen systems. During the two-year monitoring altogether four epizootics were recorded
in four different host-pathogen-habitat systems. In 2001, there was N. fresenii epizootic
observed in colonies of the black bean aphid infesting field bean (F. vulgaris). In the next
year, there was N. fresenii epizootic in the colonies of the black bean aphid infesting sugar
beet (B. vulgaris) and P. neoaphidis epizootic in the colonies of the pea aphid and the rose
grain aphid infesting field pea (P. sativum) and maize (Z. mays), respectively. The regular
monitoring of aphid colonies provided data on the seasonal development of both the host
populations and the fungal diseases. The data obtained allowed us to compare epizootic
patterns of pathogens developing in host populations with different degree of spatial
aggregation. According to DIXON /1958/, aphid species by spatial behaviour can be divided
into two fundamental groups; sedentary, clumped, aposematically coloured, toxic and/or ant
attended aphids, or mobile, more randomly dispersed, and cryptic ones. There is a number of
factors, which have been studied that may contribute to different spatial pattern of aphid
populations including the nitrogen availability /DUFFIELD et al. 1997/, density of the crop
/HONEK 1985, BASKY and HOPPER 2000/, and the effect of natural enemies like predators
/ROITBERG et al. 1979, ROY et al. 2002/, parasitoids /GOWLING and VAN EMDEN 1994, BASKY
and HOPPER 2000, HUFBAUER 2002/, and entomopathogens /CAPPUCCINO 1988, KNUDSEN et
al. 1994, BASKY and HOPPER 2000, JENSEN et al. 2001, ROY et al. 2002/. However, inversely,
the question is whether a spatial heterogeneity in aphid density may have the influence on
aphid mortality caused by a fungal pathogen. In general, the spatial pattern of biological
organism is the description of how the organism is dispersed (or disperses itself) in space
/BINNS et al. 2000b/. Aphid species with different spatial patterns are likely to differ in their
vulnerability to entomophthoralean infection /CAPPUCCINO 1988/. To date little information
can be found in the literature concerning the spatial patterns of fungus-killed aphids. FENG
and NOWIERSKI /1992a/ studied spatial patterns for four cereal aphid species killed by
entomophthoralean fungi and hymenopterous parasitoids in spring wheat in the USA. Based
on direct counts of live and fungus-killed aphids obtained during the sampling occasions, we
evaluated the spatial distribution of aphids and fungal diseases within a host plant habitat to
129
determine spatial patterns. For the three aphid species optimal sample size curves for aphid
cadavers were also developed.
Seasonal dynamics of the tree aphid species on their host plants and entomophthoralean
infection dynamics are shown in Figures 29, 30, 31, and 32. Data to plot those graphs of
Figure 29. Seasonal abundance of live Acyrthosiphon pisum and those infected by P. neoaphidis per
45 plants of field pea grown in D. Malanta in 2002
1000
Number of aphids = Log (1+x)
100
10
1
9.4.
16.4.
25.4.
2.5.
9.5.
14.5.
21.5.
28.5.
4.6.
12.6.
19.6.
26.6.
Date
living aphids P. neoaphidis
Figure 30. Seasonal abundance of live Metopolophium dirhodum and those infected by P. neoaphidis
per 45 plants of maize grown in D. Malanta in 2002
10000
1000
100
10
1
28.5.
4.6.
12.6.
19.6.
26.6.
3.7.
8.7.
Date
living aphids P. neoaphidis
130
Figure 31. Seasonal abundance of live Aphis fabae and those infected by N. fresenii per 45 plants of
field bean grown in D. Malanta in 2001
100000
10000
1000
100
10
1
2.5.
7.5.
14.5.
21.5.
31.5.
4.6.
12.6.
Date
living aphids N. fresenii
Figure 32. Seasonal abundance of live Aphis fabae and those infected by N. fresenii per 45 plants of
sugar beet grown in D. Malanta in 2002
10000
1000
100
10
1
21.5.
28.5.
4.6.
12.6.
19.6.
26.6.
3.7.
Date
living aphids N. fresenii
131
seasonal abundances are derived from Tables 56, 63, 65, and 66 (see appendix). Descriptive
analyses of spatial distribution of these aphid species are presented in the Tables 11, 12, 13,
and 14. In 2001 the first individuals of the black bean aphid, migrantes alatae, were recorded
on field bean at the beginning of May, soon after the crop had emerged. Population density
was low during the first half of May, increased during the second half of month when the crop
was flowering and peaked at early June. After the peak, the aphid abundance decreased
significantly on the following sampling date. This trend continued till a crop cutting on 23
June. One pathogenic species, N. fresenii, was identified in the aphid colonies on field bean in
2001. The infection established itself in the population at the beginning of June. At the peak
of aphid abundance a proportion of plants with a fungal infection reached 6.67% and the
disease development advanced progressively. On the following sampling occasion the
proportion of infected aphids in the population elevated significantly (χ2 = 136.61, P < 0.01).
The pathogen showed its great potential to develop disease to epizootic level, but cutting of
the crop interrupted the disease progress in the population. Nevertheless, on the final sampling
day, on 12 June, the entomophthoralean infection was already recorded on 26.67% of plants
what indicated a quick disease progress. The first infected aphids in the plot appeared when
the infestation level of the crop reached 9%. It was at the beginning of aphid arrival when all
the aphids recorded were of alate form. They colonised plants individually to settle the crop
by giving birth to apteral generations. At that time the infection was probably introduced to
the population by alatae, however, it apparently did not spread out since no new cases of
disease were found during the next two sampling dates, or the disease was not detectable by
the sampling method. The infection re-appeared after two weeks when about 80% of plants
were infested with aphids. Aphids in middle-sized colonies (6-50 or 51-150 aphids) were only
infected by the pathogen. After 4 June the infection had been established well within the host
Table 11. Analysis of spatial distribution of the black bean aphid and entomophthoralean
disease (N. fresenii) on field bean at the locality of D. Malanta in 2001
Date of sampling 2.5. 7.5. 14.5. 21.5. 31.5. 4.6. 12.6.
Aphid incidence (%) 8.89 33.33 48.89 77.78 95.56 75.56 71.11
Fungus prevalence (%) 2.22 0 0 4.44 6.67 17.78 26.67
1-5 NPC 4 11 13 5 8 7 4
Categories of infestation level
aphids / RPC (%) 100.00 73.33 61.90 14.29 18.60 20.59 12.50
plant RPI (%) 25.00 0 0 0 25.00 0 25.00
6-50 NPC 0 4 8 18 12 14 18
aphids / RPC (%) 0 26.67 38.10 51.43 27.91 41.18 56.25
plant RPI (%) 0 0 0 5.56 0 14.29 44.44
51-150 NPC 0 0 0 11 7 1 5
aphids / RPC (%) 0 0 0 31.43 16.28 2.94 15.63
plant RPI (%) 0 0 0 9.09 0 100.00 60.00
150 < NPC 0 0 0 1 16 12 5
aphids / RPC (%) 0 0 0 2.86 37.21 35.29 15.63
plant RPI (%) 0 0 0 0 6.25 41.67 0
___________________________________________________________________________
Aphid incidence – relative proportion of infested SU in the crop (number of infested SU/45x100), Fungus
prevalence – relative proportion of SU where the entomophthoralean infection was recorded (number of SU
with infection/45x100), NPC - number of SU with aphid incidence in particular category of infestation level,
RPC - relative proportion of SU of particular aphid infestation category (NPC/45x100), RPI – relative
proportion of SU with entomophthoralean infection within particular category (number of SU with
entomophthoralean infection within particular category / NPC of particular infestation category x 100)
132
colonies and the disease-positive plants were mostly infested at a level of 6-50 or 51-150
aphids.
In 2002 the black bean aphid appeared on sugar beet at the end of May and the aphid
abundance was increasing during the first half of June. After reaching a peak on 19 June, the
density dropped sharply and did not recover till harvest in the autumn. At maximum
abundance the aphid occupied 80.0% of plants investigated. N. fresenii and P. neoaphidis
infected the black bean aphid in 2002, however, the first one was the only species with the
epizootic force. The infection initiation on 4 June followed by rapid and effective distribution
of the disease within aphid colonies resulted in spectacular epizootics. Aphid mortality
increased significantly within a week, from 3.29% on 12 June to 57.54% of the population on
19 June (χ2 = 808.88, P < 0.01). At peak of abundance of mycosed aphids on 26 June a
proportion of plants with infected aphids reached 55.55% and a mortality level reached
92.93%. In the second half of June, besides sporulating cadavers, aphids filled with resting
spores were also recorded on sugar beet. Towards the end of aphid infestation the proportion
of cadavers with resting spores was increasing in relation to sporulating cadavers. Fungus-
killed aphids were first recorded on sugar beet when 33% of plants were aphid-infested. The
infection started in small colonies (1-5 aphids), which probably became foci of disease, and
with increasing aphid colonisation the infection simultaneously spread out up to 70% of
plants investigated. Majority of the infection-positive plants had a level of infestation of 6-50
or 51-150 aphids.
M. dirhodum, the rose grain aphid, appeared to be a dominant species found on maize in 2002
with up to 60 aphids per SU at population peak. Two other aphid species, R. padi and S.
avenae were also found infesting the maize plot though in low numbers not exceeding 25
aphids per plant for R. padi and with only two alate individuals of S. avenae recorded.
Initially, on 28 May, a mean population density was 11.40 aphids per SU and it reached a
maximum of 58.64 aphids per SU within two weeks. Clearly, P. neoaphidis was the
epizootic-causing agent in the aphid colonies during this season, with only two other
entomophthoralean fungi recorded, E. planchoniana and N. fresenii. E. planchoniana was
only identified from 1 and N. fresenii from 2 aphid cadavers. Sampling on 4 June indicated
that P. neoaphidis began to be active within the aphid population, although at a low level.
Infection by the fungus appeared on maize when the aphid population had well established
itself and a level of plant infestation reached 98%. The fungal mortality increased
Table 12. Analysis of spatial distribution of the black bean aphid and entomophthoralean disease (N.
fresenii) on sugar beet at the locality of D. Malanta in 2002
Date of sampling 21.5. 28.5. 4.6. 12.6. 19.6. 26.6. 3.7.
Aphid incidence (%) 8.89 24.44 33.33 71.11 80.00 6.67 0
Fungus prevalence (%) 0 0 4.44 6.67 68.89 55.55 24.44
1-5 NPC 4 8 9 8 6 0 0
Categories of infestation
aphids / RPC (%) 100.00 72.73 60.00 25.00 16.67 0 0

plant RPI (%) 0 0 22.22 12.50 33.33 0 0
6-50 NPC 0 3 5 19 22 0 0
level
aphids / RPC (%) 0 27.27 33.33 59.38 61.11 0 0

plant RPI (%) 0 0 0 5.26 90.91 0 0
51-150 NPC 0 0 1 5 8 2 0
aphids / RPC (%) 0 0 6.67 15.62 22.22 100.00 0
plant RPI (%) 0 0 0 20.00 100.00 100.00 0
___________________________________________________________________________
NPC, RPC, RPI – see Table 11
133
significantly from 0.22% on 4 June to 29.62% of the population on 12 June (χ2 = 58.33, P <
0.01). Over the next 14 days this level increased to 87.11%. The killed aphids were mostly
recorded on plants with infestation of 6-50 and 51-150 aphids and this trend carried on. It is of
interest that as early as the third sampling date, the disease had been spread to about 90% of
plants investigated. This rapid and intense expansion of disease was not observed for N.
fresenii, though infection on sugar beet had alike progress.
Table 13. Analysis of spatial distribution of M. dirhodum and entomophthoralean disease (P.
neoaphidis) on maize at the locality of D. Malanta in 2002
Date of sampling 28.5. 4.6. 12.6. 19.6. 26.6. 3.7. 8.7.

Aphid incidence (%) 64.44 97.78 100.00 100.00 100.00 97.78 62.22
Fungus prevalence (%) 0 4.44 86.67 100.00 100.00 95.56 100.00
1-5 NPC 6 13 1 0 1 13 19
aphids / RPC (%) 13.33 28.89 2.22 0 2.22 28.89 42.22

plant RPI (%) 0 0 100.00 0 100.00 100.00 100.00
6-50 NPC 22 26 24 18 17 29 9
level
aphids / RPC (%) 48.89 57.78 53.33 40.00 37.78 64.44 20.00
plant RPI (%) 0 3.85 83.33 100.00 100.00 93.10 100.00
51-150 NPC 1 5 20 27 28 1 0
aphids / RPC (%) 2.22 11.11 44.44 60.00 62.22 2.22 0
plant RPI (%) 0 20.00 90.00 100.00 100.00 100.00 0
___________________________________________________________________________
In 2002 the pea aphid was first detected on field pea on 9 April with zero records on the
following two observations. The aphid abundance was very low till 14 May (< 2.4 aphids per
SU, incidence level < 2.5%), but increased significantly in the following week and peaked on
28 May (> 12 aphids per SU). Notably, P. neoaphidis was the only significant pathogen
recorded from collected cadavers during 2002. Infection by C. obscurus was also observed.
The first sign of fungal activity was observed on 14 May, though the mortality did not exceed
1%. The highest aphid mortality due to P. neoaphidis (40.60%) was recorded on 19 June.
This level, however, declined significantly to 20.81% on 26 June (χ2 = 18.53, P < 0.01). The
infection was recorded for the first time when 44% of plants were infested with aphids and it
was observed within small-sized colonies (1-5 aphids). Later the infection dispersed itself to
80% of infested plants, though the progress was not as dynamic as on maize. Majority of
disease-positive plants were infested in the level of 6-50 aphids. While the infestation of
plants was relatively stable from 21 May to 26 June, the fungus prevalence varied from 10 to
80%.
It can be generalised that A. fabae aggregated when colonised the host plants and
subsequent apteral generations were relatively sedentary, resulting in extremely high numbers
of aphids per host plant (e.g. > 330 individuals per SU at the population peak on field bean).
On the contrary, M. dirhodum and A. pisum had more sparse spatial distribution and infested
plants more evenly. These two aphid species were also typical of infesting a great number of
host plants within the crops in relatively short time. After reaching a maximum incidence in
the crop, when highest number of plants was infested, M. dirhodum and A. pisum continued to
sustain this level of incidence practically until the colonies were observed within the crops
(Tables 13 and 14). However, in case of A. fabae after reaching a peak of incidence,
population was decreasing to nearly zero (Table 12). It was not observed on field bean since
mowing of the crop untimely broke off the population development. Assorting the sample
134
Table 14. Analysis of spatial distribution of the pea aphid and entomophthoralean disease (P.
neoaphidis) on pea plots at the locality of D. Malanta in 2002
Date of sampling 2.5. 9.5 14.5. 21.5. 28.5. 4.6. 12.6. 19.6. 26.6.
Aphid incidence (%) 2.22 26.67 44.44 82.22 93.33 75.56 91.11 93.33 91.11
Fungus prevalence (%) 0 0 2.22 15.56 11.11 28.89 35.56 82.22 62.22
1-5 NPC 0 10 13 17 14 9 13 5 15
aphids / RPC (%) 0 22.22 28.89 37.78 31.11 20.00 28.89 11.11 33.33
plant RPI (%) 0 0 7.69 11.76 7.14 0 0 60.00 46.67
6-50 NPC 1 2 7 20 28 24 28 37 27
level
aphids / RPC (%) 2.22 4.44 15.56 44.44 62.22 53.33 62.22 82.22 60.00
plant RPI (%) 0 0 0 25.00 14.29 50.00 57.14 94.59 77.78
51-150 NPC 0 0 0 0 0 1 0 0 0
aphids / RPC (%) 0 0 0 0 0 2.22 0 0 0
plant RPI (%) 0 0 0 0 0 100.0 0 0 0
___________________________________________________________________________
units by level of aphid infestation we observed that, in general, prevalence of

entomophthoralean infection grew with infestation of sample units with aphids. This
observation confirms the general knowledge about a host density dependence of the diseases
/STEINHAUS 1954, WATANABE 1987/, which was presented by several authors in aphid
populations /e.g. WILDING and PERRY 1980, FENG et al. 1991/. For example, aphids in larger
colonies on goldenrod stems are more vulnerable to N. fresenii infection than aphids in
smaller ones and even the infection appears sooner in the larger colonies /CAPPUCCINO 1988/.
It was reported that aphids themselves might serve as important inoculum spreading factors
and they might be responsible for introduction of primary infection to colonies /WILDING and
PERRY 1980, ČUDARE 1990/. On field bean, sugar beet, and field pea we found first infected
aphids in small recently established colonies. This might indicate that the disease was
introduced to the plots by founders of colonies, though alate cadavers were only detected in
these colonies from field bean. The threshold aphid density at which the pathogens start to be
an effective reducing factor is generally high and epizootics in populations usually occur at
high host densities /SIVČEV 1991, PELL et al. 2001/. During our observations the notable
increase in fungal activity started after a peak of aphid abundance. The high level of aphid
mortality recorded during decreasing stage of population dynamics may be attributed, in all
probability, to the migration of healthy aphids from plants of lower nutritional value while
cadavers remained attached to the plants. Since the Entomophthorales became active when
populations were decreasing it could be assumed that physiological state of aphids was more
important for epizootic development than the high numbers of hosts. We observed that despite
the mortality of A. fabae on sugar beet due to fungal infection was great, the prevalence of
infection, relative proportion of infection positive plants, did not exceed 70%. On the other
hand in case of M. dirhodum colonising maize the prevalence of disease reached quickly
100% of plants. The fast spread of P. neoaphidis infection to all plants of maize investigated
(within 14 days) may be attributed to high crop infestation by the pest (97.78 aphids per SU)
when first infection appeared. Lower prevalence of disease was recorded on field bean, while
that on pea was comparable to infection prevalence on maize. Differences in aphid behaviour
may explain this phenomenon. A. fabae was more sedentary and aggregated on the plants and
so, while the aggregation increases the likelihood of live aphids to come into contact with
sporulating cadavers within colonies, a dissemination of pathogen among the host colonies
due to the low aphid mobility was limited. Mobility of aphids is also important parameter that
135
can influence a host coming into contact with inoculum. This is the case predominantly for N.
fresenii, which infective conidia, capilliconidia, are not forcibly ejected and they remain on
the leaf surface until picked up by passing hosts. However, the high host densities (e.g. A.
fabae), can probably offset the low mobility and thus inoculation through direct contact with
killed individuals prevail over random picking up of conidia.
In spite of that pea and maize plots were merely several metres away the P. neoaphidis
infection in pea aphid and rose grain aphid colonies had different dynamics. In pea aphid
population the infection appeared three weeks earlier and it had been well established when
first infected M. dirhodum were observed. It could not be ruled out that sporulating pea aphid
cadavers were a source of inoculum for M. dirhodum infection. Since infection route for
Entomophthorales is through the integument, via direct contact of pathogen with host, a
horizontal transmission is the principal mode of fungal spreading within and among aphid
colonies /WILDING and PERRY 1980, PELL et al. 2001/. Transmission of infection among
different aphid species has been observed /STEENBERG and EILENBERG 1995, SHAH et al.
2004/ and successful transmission of entomophthoralean disease between different aphid
species was carried out in this research as well (see section 5.3.3). One could assume that
inoculum from sporulating pea aphids might have a supporting effect on the dynamic progress
of disease in the nearby crop, although other favourable stimuli had to be in attendance.
Edge effects, which are commonly determined in aphid spatial distribution /DEAN 1973,
RUGGLE and HOLST 1995, WINDER et al. 1998, 1999/, were not evaluated in this study
because of small size of plots incorporated into research. Edge effects may be due to a number
of factors and may have several forms /DEAN 1973, RUGGLE and HOLST 1995/.
We used index of aggregation (λ) to estimate aggregation for live and fungus-killed aphids
within the populations at each sampling date. Tables 15-18 show the index of aggregation for
all the aphid populations. It was observed that aphid distribution tended to be highly
aggregated at low mean densities per SU. In contrast, the distribution tended to be more
random at high host densities and when a high number of plants tended to be infested. This
phenomenon is with agreement with observations of PALUMBO et al. /2000/ who studied a
field distribution of aphids on head lettuce crops. Using regression analysis we tested
relationship between the variables of aggregation index (λ) and proportion of plants with
aphids and between variables of aphid mortality and aggregation index (λ) of live aphids
(Table 19). The analysis confirmed strong negative relationship between aggregation of
aphids and plants infested with aphids, though the correlation was only significant (P < 0.05)
for A. fabae on field bean and A. fabae on sugar beet. Aphid mortality was medium or strong
positively dependent upon aphid aggregation for all the aphid species except for A. pisum.
However, the correlation was not statistically significant for either aphid species (P < 0.05).
A. pisum astonishingly shoved negative correlation between aphid mortality and spatial
aggregation. The reason for this is unknown, though this may relate with more random spatial
distribution within the plot and greater aphid mobility among host plant and thus allowing
disease dispersal in the crop.
Spatial distribution expressed by index of aggregation (λ) was confirmed by using a χ2-test
for departure from randomness (Tables 15-18). Nearly all values of χ2 for A. fabae (live and
fungus-killed individuals) irrespective of host plant suggested an aggregated spatial pattern
and substantial departure from randomness (P > 0.05). However, in case of A. pisum the χ2
values were only significantly larger than the tabled values at the 0.05 level for live aphids
during the first half of population development, suggesting a spatial aggregation. For M.
dirhodum values of χ2 suggested an aggregated spatial pattern at the beginning and the end of
population. The χ2 values for cadavers of A. pisum were significant on sampling occasions 3-7
and not at the end of population development on occasion 8 and 9. In case of M. dirhodum
cadavers, spatial pattern was present at the beginning and the end of population development.
136
Table 15. Summary statistics for live A. fabae and fungus-killed aphid data collected in the field
bean plot during the 2001 growing season at D. Malanta ( x – arithmetic aphid density per SU, s2 –
variance, λ – index of aggregation, χ2 – χ square test at P = 0.05)
Date of Live aphids Aphids killed by N. fresenii
sampling x s 2
λ χ 2
x s2 λ χ2
2.5. 0.13 0.25 0.56 650.89* 0.02 0.02 1.00 2200*
7.5. 1.27 13.15 0.43 358.73* 0 0 - -
14.5. 4.53 98.66 0.33 211.54* 0 0 - -
21.5. 39.78 2871.49 0.20 79.84* 0.04 0.04 0.70 1100*
31.5. 344.87 283273.75 0.23 104.80* 0.22 1.18 0.73 1072.73*
4.6. 220.40 206332.47 0.31 186.89* 2.76 68.73 0.45 396.99*
12.6. 57.87 11549.25 0.28 151.74* 4.64 125.19 0.36 255.85*
____________________________________
* significant, P = 0.05
Table 16. Summary statistics for live A. fabae and fungus-killed aphid data collected in the sugar beet
plot during the 2002 growing season at D. Malanta ( x – arithmetic aphid density per SU, s2 –
Date of Live aphids Aphids killed by N. fresenii

sampling x s2
λ χ
2
x s2 λ χ2
21.5. 0.09 0.08 0.48 434.57* 0 0 - -
28.5. 1.47 19.89 0.45 405.00* 0 0 - -
4.6. 7.53 151.07 0.24 117.23* 0.09 0.17 0.70 923.46*
12.6. 19.44 692.07 0.20 80.58* 0.67 10.73 0.73 1051.73*
19.6. 26.31 873.81 0.17 55.54 34.29 2678.30 0.22 100.23*
26.6. 2.78 40.40 0.34 230.01* 36.13 6254.44 0.33 210.82*
3.7. 0 0 - - 7.69 306.04 0.34 227.71*
____________________________________
Table 17. Summary statistics for live A. pisum and fungus-killed aphid data collected in the pea plot
during the 2002 growing season at D. Malanta ( x – arithmetic aphid density per SU, s2 – variance, λ –
index of aggregation, χ2 – χ square test at P = 0.05)
Date of Live aphids Aphids killed by P. neoaphidis

sampling x s 2
λ χ 2
x s2 λ χ2
2.5. 0.29 3.76 1.00 1967.18* 0 0 - -
9.5. 0.49 1.62 0.39 296.88* 0 0 - -
14.5. 2.38 24.01 0.31 186.51* 0.02 0.02 1.00 2200*
21.5. 10.44 148.75 0.17 60.05* 0.16 0.13 0.35 223.44*
28.5. 12.49 109.26 0.12 30.82 0.11 0.10 0.45 363.64*
4.6. 10.91 172.32 0.18 63.70* 0.84 3.36 0.32 209.52*
12.6. 5.36 31.87 0.16 48.81 0.44 0.75 0.29 170.45*
19.6. 10.67 93.91 0.14 36.29 7.31 37.95 0.13 31.25
26.6. 8.71 56.35 0.13 32.68 2.29 6.21 0.16 52.10
____________________________________
137
Table 18. Summary statistics for live M. dirhodum and fungus-killed aphid data collected in the maize
plot during the 2002 growing season at D. Malanta ( x – arithmetic aphid density per SU, s2 –
Date of Live aphids Aphids killed by P. neoaphidis

sampling x s2 λ χ 2
x s2 λ χ2
28.5. 11.27 217.97 0.20 75.51* 0 0 - -
4.6. 20.13 624.57 0.19 67.82* 0.04 0.04 0.70 1100*
12.6. 44.04 740.73 0.09 16.80 4.56 16.30 0.13 34.49
19.6. 58.64 1335.92 0.09 17.09 39.07 1135.02 0.13 32.72
26.6. 6.58 42.29 0.15 42.98 44.47 664.30 0.09 14.78
3.7. 0.36 0.73 0.36 247.84* 11.44 155.84 0.16 52.39
8.7. 0.16 0.23 0.45 395.31* 2.76 15.73 0.21 90.86*
____________________________________
Table 19. Parameters of regression analyses for aphid aggregation, plant infestation, and aphid
mortality from four seasonal dynamics monitored at D. Malanta during 2001-2002
Independent x dependent variable Regression equation Corel. coeff. F P
A. fabae, field bean, 2001:
λ (live aphids) x plants with aphids (%) y = 0.564 – 0.004x - 0.940 37.834 0.0017*
aphid mortality (%) x λ (live aphids) y = -6.345 + 33.678 x + 0.456 0.524 0.5442
A. fabae, sugar beet, 2002:
λ (live aphids) x plants with aphids (%) y = 0.443 – 0.004x - 0.828 8.697 0.0420*
aphid mortality (%) x λ (live aphids) y = -41.103 + 335.792x + 0.559 0.908 0.4412
A. pisum, field pea, 2001:
λ (live aphids) x plants with aphids (%) y = 0.548 – 0.005x - 0.623 0.433 0.0733
aphid mortality (%) x λ (live aphids) y = 26.750 – 88.628x - 0.389 0.889 0.3891
M. dirhodum, maize, 2002:
λ (live aphids) x plants with aphids (%) y = 0.679 – 0.005x - 0.612 2.999 0.1439
aphid mortality (%) x λ (live aphids) y = -17.861 + 183.152x + 0.730 4.575 0.0992
________________________________________________________
F – test characteristic, P – significance for F, * significant at P = 0.05
Taylor’s power law /TAYLOR 1961, 1971/ was used to model a relationship between mean
densities (per sample unit) and variances for both living and fungus-killed aphids of all the
aphid species. The results of fitting Taylor’s model to data for each aphid populations are
listed in the Table 20. The power law described well the relationship between the log-
transformed means and variances for both, live and fungus-killed aphids. The resulting
regression equations had high coefficients of determination (r2) indicating a good fit of the
model. The coefficients of determination were over 0.950 for live aphids (P < 0.001) and
regressions involving fungus-killed aphids gave similar values. Comparable results of good fit
to the model were also reported by FENG and NOWIERSKI /1992a,b/ for cereal aphids, but their
coefficients of determination were lower for fungus-killed aphids. Ordinarily, Taylor’s power
law can be estimated well only if data are available from a wide range of pest densities, and if
the estimated means and variances are reasonably precise. A general rule is that at least 40
138
sample observations to estimate each sample mean and variance are necessary /BINNS et al.
2000a/, although smaller numbers have also been successfully used /e.g. TAYLOR et al. 1988/.
Table 20. Taylor’s power law analysis; regression of log variance (s2) and log mean ( x ) for live aphid
(L) and aphids killed by P. neoaphidis (PN) or N. fresenii (NF), (log a – intercept, b – slope, t – t-test
of slope, r2 – coefficient of determination, SE – standard error, P – P value of the regression)
log a b (SE) t (stat) t (tab) r2 No. obs. P

A. pisum, L 0.879 1.100 (0.124) 8.848 3.499 (P=0.01) 0.958 9 < 0.0001
pea, 2002 PN 0.389 1.329 (0.084) 15.909 4.032 (P=0.01) 0.990 7 < 0.0001
M. dirhodum, L 0.563 1.503 (0.077) 19.657 4.032 (P=0.01) 0.994 7 < 0.0001
maize, 2002 PN 0.552 1.442 (0.079) 18.302 4.604 (P=0.01) 0.994 6 < 0.0001
A. fabae, L 0.898 1.788 (0.058) 31.020 4.032 (P=0.01) 0.997 7 < 0.0001
bean, 2001 NF 1.053 1.690 (0.077) 22.024 6.925 (P=0.01) 0.998 4 0.0021
A. fabae, L 0.773 1.629 (0.088) 18.580 4.604 (P=0.01) 0.994 6 < 0.0001
s. beet, 2002 NF 1.087 1.645 (0.092) 17.944 5.841 (P=0.01) 0.995 5 0.0004
Estimates of slope b were significantly > 1.00 (P < 0.01) for all live aphids varying in
degree with the aphid species. This indicates according to TAYLOR /1961, 1971/ that the
spatial patterns were all aggregated. The slopes also significantly exceeded 1.00 for fungus-
killed aphids (P < 0.01). The parameter b is usually regarded as an index of aggregation and is
species-specific, while intercept a depends primarily on the sampling method /TAYLOR 1961/.
It is often possible to show differences in a or b due to the effects of plant cultivar or variety,
the presence or absence of natural enemies, pesticide application or geographical location
/BINNS et al. 2000a/. According to the slopes, the distribution of live and fungus-killed aphids
appeared to be aggregated. Fungus-killed aphids were more aggregated than live aphids in
case of A. pisum, however, this was not true for M. dirhodum on maize and A. fabae on bean.
The slopes for live and fungus-killed aphids of A. fabae on sugar beet were close. Statistical
tests for homogeneity of the slopes (Table 21) revealed that differences in aggregation
between live and fungus-killed aphids were not significant. This is in consistence with
observations of FENG and NOWIERSKI /1992a/ who also found no significant differences
between slopes of living cereal aphids and slopes of fungus-killed aphids. This suggests that
the distribution of the aphids killed by entomophthoralean pathogens simply reflects the
spatial pattern of the aphid populations. Variation among the spatial aggregation of
populations of live aphids appeared. The populations were aggregated in the following order
A. pisum on pea < M. dirhodum on maize < A. fabae on sugar beet < A. fabae on field bean.
M. dirhodum and A. pisum colonised host plants singly and were more mobile. These
behaviours resulted in more random spatial pattern. Homogeneity of spatial patterns of live
Table 21. Homogeneity of regression slopes from Taylor’s power law between fungus-killed and
live aphids (H 0 : b fungus-killed = b live )
Fungus-killed vs. live aphids b f.-killed b live SE f.-killed SE live df t
A. pisum, pea, 2002 1.329 1.100 0.084 0.124 14 -1.529
M. dirhodum, maize, 2002 1.442 1.503 0.079 0.077 11 0.553
A. fabae, bean, 2001 1.690 1.788 0.077 0.058 9 1.017
A. fabae, s. beet, 2002 1.645 1.629 0.092 0.088 9 -0.126
139
Table 22. Homogeneity of regression slopes from Taylor’s power law among populations of live
aphid at Dolná Malanta (H 0 : b 1 = b 2 )
Population 1 vs. population 2 b1 b2 SE 1 SE 2 df t

A. fabae, bean, 2001 vs.
1.788 1.100 0.058 0.124 14 5.026**
A. pisum, pea, 2002
1.788 1.503 0.058 0.077 12 2.956*
M. dirhodum, maize, 2002
1.788 1.629 0.058 0.088 11 1.509
A. fabae, s. beet, 2002
A. fabae, s. beet, 2002 vs.
1.629 1.100 0.088 0.124 13 3.479**
A. pisum, pea, 2002
1.629 1.503 0.088 0.077 11 1.078
M. dirhodum, maize, 2002 vs.
1.503 1.100 0.077 0.124 14 2.761*
A. pisum, pea, 2002
___________________________________________
* significant at P = 0.05, ** significant at P = 0.01
Table 23. Homogeneity of regression slopes from Taylor’s power law among numbers of fungus-
killed aphids in populations at Dolná Malanta (H 0 : b 1 = b 2 )
Population 1 vs. population 2 b1 b2 SE 1 SE 2 df t

1.690 1.329 0.077 0.084 9 3.168*
A. pisum, pea, 2002
1.690 1.442 0.077 0.079 8 2.248
1.690 1.645 0.077 0.092 7 0.375
A. fabae, s. beet, 2002
1.645 1.329 0.092 0.084 10 2.536*
A. pisum, pea, 2002
1.645 1.442 0.092 0.079 9 1.674
M. dirhodum, maize, 2002 vs.
1.442 1.329 0.079 0.084 11 0.980
A. pisum, pea, 2002
___________________________________________
* significant at P = 0.05
aphids is shown in Table 22. Colonies of A. fabae on field bean were significantly more
clumped than those of A. pisum on pea, M. dirhodum on maize, colonies of A. fabae on sugar
beet were significantly more clumped than those of A. pisum on pea, and finally colonies of
M. dirhodum on maize were significantly more aggregated than those of A. pisum on pea.
Certain degree of variation among Taylor’s slopes for fungus-killed aphids was also observed.
The aggregation of cadavers increased in the following order A. pisum on pea < M. dirhodum
on maize < A. fabae on sugar beet < A. fabae on field bean. Significant differences in spatial
aggregation was only between A. fabae killed by N. fresenii on field bean and A. pisum killed
140
by P. neoaphidis on pea, as well as, between A. fabae killed by N. fresenii on sugar beet and
A. pisum killed by P. neoaphidis on pea (Table 23).
The functional relationship between the proportion of SU bearing fungus-killed aphids
(P 1 ) of each aphid species and the mean density of fungus-killed aphids per sample unit is
shown in the Figure 33. The more aggregated the distribution of aphid cadavers, the more
slowly do the values of P 1 increase with the means. For instance, the P 1 ’s for A. fabae
cadavers, which were more clumped in spatial pattern than cadavers of A. pisum and M.
dirhodum, increased slowly compared with those for A. pisum and M. dirhodum cadavers.
Thus, equal prevalence of Entomophthorales in the populations of A. pisum and M. dirhodum
seems to require less time to spread out in the crops, when compared with more aggregated A.
fabae, due probably
Figure 33. Proportion of sample units (SU) bearing fungus-killed aphids (P1) predicted from the
density of killed aphids per sample unit using NDB
0,9 AP
MD
0,8
0,7
AF+BV
Pr opor tion (P1)
0,6
0,5
AF+FV
0,4
0,3
0,2
0,1
0
0,0 2,0 4,0 6,0 8,0 10,0 12,0 14,0
Mean no. of cadavers per SU
––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––-
AP – Acyrthosiphon pisum, MD – Metopolophium dirhodum, AF+FV – A. fabae on field bean, AF+BV – A.
fabae on sugar beet
to a higher mobility of the hosts. Conidia produced on fungus-killed aphids are projected only
a short distance and infect aphid hosts almost in situ. Thus, in more aggregated and sedentary
aphid colonies, like those of A. fabae, disease spreads out fast within the aphid colony,
resulting in higher mean population mortality, however, the probability to encounter a disease
positive colony in the crop is lower. This is in agreement with results of descriptive analysis
on spatial distribution for the aphids discussed above. FENG and NOWIERSKI /1992a/
compared the values of P 1 for fungus-killed cereal aphids and aphids killed by parasitoids.
They concluded that the P 1 estimates for fungus-killed aphids increased slower compared
with those for aphids killed by parasitoids. The functional relationship between the P 1 ’s and
the means provide the possibility to estimate of fungal prevalence in the crop by merely
counting the number of SU bearing the fungus-killed aphids. The modelled P 1 values
correlated quite well to the values of P 1 observed in the crops for all the populations: A.
141
pisum, M. dirhodum, and A. fabae on field bean and sugar beet (r2 = 0.981, P < 0.0001; r2 =
0.900, P < 0.05; r2 = 0.980, P < 0.05; r2 = 0.958, P < 0.05; respectively).
Estimation of mean aphid density on crops is inevitable step when determining decision
plan for chemical control of aphids. The precision of estimation is chiefly influenced by a size
of sample. To generate an optimal sample size for fixed levels of precisions, the usual
expression for the standard error of the mean is used by substituting parameters from the
Taylor’s power law /e.g. FENG and NOWIERSKI 1992, TSAI et al. 2000/. We generated the
optimal sample size for determining the mean prevalence of entomophthoralean infection in
the crops. The sample size curves for fixed levels of precision were calculated for each aphid
species and crop (Figure 34). Three confidence levels (D = 0.1, 0.3, and 0.5, which are
equivalent to 5, 15, and 25% of standard error of mean, respectively) were tested. The
relationship shows a requirement of quite large sample size to obtain relatively precise density
estimate. To estimate a mean density of fungus-killed aphids for a given level of confidence,
more samples are needed for more aggregated A. fabae cadavers than for the other two
species. For example, when D = 0.1, estimating a prevalence of infection at 10 cadavers per
SU required 539 SU for the aphid on sugar beet, and 553 SU for the aphid on field bean.
Conversely, less aggregated aphids required at the same level of precision and infection
prevalence 98 SU for M. dirhodum or 52 SU for A. pisum. It was observed that an increase in
Taylor’s slope coefficient causes an increase in sample size requirements. This characteristic
was presented by other authors as well /e.g. ELLIOTT 1977, FENG and NOWIERSKI 1992a,b/.
Besides the slope, intercept may also influence the estimated sample size /FENG and
NOWIERSKI 1992a/. Reducing the precision level may markedly decrease the number of SU
required for estimates at the same level of fungal prevalence. For example, when D = 0.5,
Figure 34. Optimal sample size curves for fungus-killed aphids at three confidence levels of 5% (▬),
15% (▬), and 25% (▬).
2000 Sugar beet / A. fabae / N. fresenii 2000 Bean / A. fabae / N. fresenii
1500 1500
Sample size
Sample size
1000 1000
500
500
0
0
0,0 1,0 2,0 3,0 4,0 5,0
0,0 10,0 20,0 30,0
Mean no. of cadaver s per SU Mean no. of cadaver s per SU
2000 Pea / A. pisum / P. neoaphidis 2000 Maize / M. dirhodum / P. neoaphidis
1500 1500
Sample size
Sample size
1000 1000
500
500
0
0
0,0 5,0 10,0 15,0 20,0 25,0 30,0
0,0 1,0 2,0 3,0
Mean no. of cadaver s per SU Mean no. of cadaver s per SU 142
and prevalence of disease is 10 cadavers per SU sample sizes decreased to 22 SU for A. fabae
on field bean. Obviously, the choice of confidence level dramatically influences cost of time
and effort invested into a population sampling. To obtain relatively precise density estimates,
quite large sample sizes are required. Such precision in density estimates may be required for
research purpose, but it is not of practical use. Thus making a decision about an optimal
confidence level should be a compromise of precision and practicality of the work. FENG and
NOWIERSKI /1992b/ recommend for scouting purposes of cereal aphids at least a 25% error
level, however, for research purposes error levels of or below 15% are more desirable.
5.3.3 NON-CROP VEGETATION AS RESERVOIR FOR THE PATHOGENS IN LANDSCAPE WITH SPECIAL
ACCENT TO NETTLE PATCHES
Majority of detailed field studies on Entomophthorales have been focused on pestiferous

aphid species, e.g. aphids in cereals /DEAN and WILDING 1971, DEDRYVER 1983, FENG et al.
1990, 1992a, ŠTALMACHOVÁ and CAGÁŇ 2000/, the green peach aphid /ELKASSABANY et al.
1992, KISH et al. 1994/, the black bean aphid /WILDING and PERRY 1980/, or the pea aphid
/VORONINA 1971, MILNER 1982, CAGÁŇ and BARTA 2001/. However, it is believed that aphids
inhabiting non-crop vegetation in the vicinity of crops may serve as a significant reservoir for
the fungal disease. From these habitats the fungal inocula may spread out through biotic (e.g.
co-occurring insects) and abiotic (e.g. wind and rain) transmission agents and affect aphid
populations in adjacent crops. This is an important feature for development of conservation
biological control strategies /SHAH et al 2004/. Research efforts in the field of importance of
non-crop vegetation have focused mostly on the value of these sites as harbours for aphid-
specific parasitoids and predators in arable landscape /STARÝ 1983, POWELL et al. 1986a,
SCHMID 1992, NICOLI et al. 1995, LEATHER et al. 1999/, but a little attention has been devote
to study of entomopathogenic fungi /POWELL et al. 1986a, STEENBERG and EILENBERG 1995,
POPE et al. 2002/. During our extensive study on aphid-specific Entomophthorales we have
observed numerous wild plants, herbs, or trees attacked by huge amount of aphid numbers
often dying of entomophthoralean infection at different prevalence levels (see section 5.1).
These observations have evoked us to evaluate significance of that vegetation as a pathogen
reservoir in nature. In this respect, we aimed special attention to the stinging nettle, Urtica
dioica Linné, for several grounds: (1) stinging nettle is a vigorous perennial plant that often
occurs in dense stands as element of bordering flora and vegetation of field edges /SRUTEK
and TECKELMANN 1998/ with widespread distribution in central Europe /PRACH and WADE
1992/; (2) U. dioica patches serve as important primary or alternative feeding sites for many
arthropods including some beneficial natural enemies; studies on diversity and abundance of
insect fauna on U. dioica in Europe revealed 48 /DAVIS 1989/ or 55 /ZABEL and TSCHARNTKE
1998/ phytophagous and 14 predatory species /ZABEL and TSCHARNTKE 1998/; (3) out of the
phytophagous insects two monophagous aphid species, Microlophium carnosum and Aphis
urticata, can be annually encountered on stinging nettle in considerable numbers; the aphid
species often suffer from several entomophthoralean diseases /e.g. LEATHERDALE 1970,
KELLER 1991, BAŁAZY, 1993/.
The objective was primarily to determine an entomophthoralean complex associated with
the nettle aphids and then to discuss a possible role of stinging nettle patches as a pathogen
reservoir in agricultural landscape.
Two aphid species, M. carnosum and A. urticata, were recorded from the stinging nettle
during the survey. A. urticata infested nettle at the locality in 1999, 2001, and 2002, but few
nettle stalks were observed being colonised with dense colonies. Since the aphid species
seemed being scarce, the stalks inhabited with A. urticata were not evaluated in the study. The
common nettle aphid, M. carnosum, created relatively dispersed colonies and occurred on
shoots and the underside of leaves of the stinging nettle plants. Tables 67-70 (see appendix)
143
show development of abundance of the nettle aphid observed throughout the four sampling
periods and a composition of entomophthoralean complex active in the colonies. The first
aphids colonized the nettle patches during May (1998, 1999) or during April (2001, 2002).
The population density increased gradually and peaked in the second half of June (1998,
1999), in the middle or at the end of May (2002, 2001, respectively). In July the aphids
disappeared and did not re-appeared in the season. Five entomophthoralean fungal species
were identified from the common nettle aphid at the locality: P. neoaphidis, N. fresenii, N.
microlophii, E. planchoniana, and C. obscurus. Among the species, the first three ones were
major in the aphid populations and infected their host from May (beginning of April in 2001)
till July when the aphids disappeared. E. planchoniana was only identified in 2001 and C.
obscurus in 2001 and 2002. Each of these two pathogens was recorded just from one aphid
cadaver in 2001. C. obscurus cadaver was found on 4 June and the aphid killed by E.
planchoniana was observed on 23 June. C. obscurus was more active in 2002 when nearly 40
infected aphids were detected.
While composition of pathogen species was similar in all the years the infection rate and
disease development was rather different. In 1998 the infection started on 31 May when 8
individuals killed by P. neoaphidis were found. First aphids killed by Neozygites spp. were
recorded from the locality 10 days later. The entomophthoralean infections quickly
established themselves within aphid population and by 17 June, 18 days after the first killed
aphids appeared. A number of mycosed aphids peaked with infection rate of 41.29%. The
mycoses remained present in host population until the end of aphid infestation. From 19 June
aphids filled by resting spores of Neozygites spp. were present regularly in the colonies.
In 1999 the infection pattern was similar to the previous one. Killed aphids were first found
on 28 May, but the infection rate remained low until the middle of June. N. microlophii
appeared on 28 May followed by P. neoaphidis after 3 days and N. fresenii after a week.
Following a peak on 30 June a number of fungus-killed aphids diminished to a half and no
living or dead aphids were recorded after 2 July. Aphids filled by resting spores appeared
from 28 June.
Contrary to the years 1998 and 1999, in 2001 the entomophthoralean infection started
already at the beginning of April. 10 P. neoaphidis cadavers per investigated stalks were
noted on 3 April. Infection of the aphid continued at low level till the end of May and reached
maximum of 140 cadavers per stalks on 12 June. P. neoaphidis was dominant pathogen
whereas Neozygites spp. infected just a few aphids in late May and early June. No Neozygites
resting spores were observed. Out of 37 Neozygites cadavers observed through the season 2
aphids were only killed by N. microlophii.
In 2002, the first sign of infection in the population was recorded in the middle of May.
During the season three fungi were identified P. neoaphidis, N. fresenii, and C. obscurus,
however, prevalence of infection was low. Two aphids killed by N. fresenii were only
observed. On the other hand higher prevalence of C. obscurus was recorded.
P. neoaphidis, N. fresenii and N. microlophii were involved into transmission experiments
to evaluate a chance for cross-infection among aphid colonies of various species. A close host
specificity of pathogen N. microlophii to species Microlophium carnosum was underlined by
the bioassay when none of the treated aphid species succumbed to the mycosis. Further two
entomophthoralean fungi exhibited ability to infect several aphid species at different mortality
level. The average proportion of aphids exhibiting mycosis (i.e. death followed by
sporulation) after direct inoculation with spores produced from P. neoaphidis-killed aphids
ranged between 0 and 33.33% depending on aphid species and with spores from N. fresenii-
killed aphids ranged between 0 and 15% (Table 24).
144
Table 24. Results of P. neoaphidis and N. fresenii transmission experiment represented with values of
average relative mortality of tested aphids after exposition to spores from killed M. carnosum (number
of mycosed aphids/number of aphids tested x 100), SE – standard error of the mean
Average mortality ± SE
Pathogen Aphid species tested Original host of aphid
(%)
Aphis fabae Scopoli Faba vulgaris Moench. 33.33 ± 11.67
Macrosiphum rosae (Linné) Rosa canina Linné 20.00 ± 5.00
neoaphidis
Hyalopterus pruni (Goeffroy) Prunus spinosa Linné 0

Pandora
Rhopalosiphum maidis (Fitch) Hordeum vulgare Linné 15.00 ± 7.64

Metopolophium dirhodum (Walker) Zea mays Linné 33.33 ± 11.67
Acyrthosiphon pisum (Harris) Pisum sativum L. 31.67 ± 11.67
Aphis fabae Scopoli Faba vulgaris Moench. 15.00 ± 2.89
Macrosiphum rosae (Linné) Rosa canina Linné 0
Neozygites
Hyalopterus pruni (Goeffroy) Prunus spinosa Linné 0

fresenii
Rhopalosiphum maidis (Fitch) Hordeum vulgare Linné 8.33 ± 1.67

Metopolophium dirhodum (Walker) Zea mays Linné 6.67 ± 4.41
Acyrthosiphon pisum (Harris) Pisum sativum Linné 5.00 ± 0.00
M. carnosum is rather common aphid species of stinging nettle in central Europe. It is often
found in forest edges, parks, roadsides, fallow land in fields, and waste places in urban
environment with its co-occurring aphid species A. urticata, but both aphids are seldom found
together on the same plant /STARÝ 1983/. In the present study a regular occurrence of the
common nettle aphid was documented during the 4-year monitoring. In central Europe the
aphid is usually present on U. dioica from early June to the end of August with a peak of
abundance from the second half of June to about mid-July /STARÝ 1983/. Under conditions of
southern Slovakia, where the study area was situated, the population development of the aphid
was advanced in about 2-3 weeks and in autumn the aphid did not appear. In southern
England M. carnosum reaches high population densities in late spring and the abundance
drops to very low level in mid to late summer. Moreover, in early autumn the population may
be also observed in low prevalence /MÜLLER and GODFRAY 1997/.
Populations of M. carnosum have been observed that are susceptible to entomophthoralean
infections in several countries. P. neoaphidis, E. planchoniana, C. obscurus, N. fresenii, N.
microlophii, C. coronatus, and Z. phalloides have been reported from the aphid species up to
now /e.g. BATKO 1966a, LEATHERDALE 1970, THOIZON 1970, MIĘTKIEVSKY and VAN DER
GEEST 1985, KELLER 1987a, 1991, BAŁAZY 1993/. The first five species were observed
during our investigation in Slovakia. P. neoaphidis, E. planchoniana, C. obscurus, and N.
fresenii are widespread aphid pathogens and the host ranges of the species are considered
wide, since the pathogenic fungi have been found on a large number of aphid species /e.g.
THOIZON 1970, LATGÉ and PAPIEROK 1988, KELLER 1987a, 1991, BAŁAZY 1993/. P.
neoaphidis, as the most important aphid pathogenic fungus, regularly causes epizootics
among aphid populations in field crops /e.g. KELLER 1991, BAŁAZY 1993/. In Slovakia the
pathogen was present in the aphid colonies every year with abundance at great levels what
corresponds with its general distribution and high pathogenicity. Rather high effectiveness
and wide specificity confirmed by successful horizontal transmission place the fungus
unquestionably among to the most perspective species for aphid control. Though the fungus
presumably does not form resting spores /BAŁAZY 1993/ and way of overwintering as well as
a form by which is preserved in nature are unknown, or a matter of discussion /Nielsen et al.
2003/, its role as reduction factor of aphids is important because it may appear already during
early spring when aphid populations are just establishing in crops. N. fresenii, further common
aphid-specific pathogen /KELLER 1997/, was observed at different levels of prevalence within
145
the aphid populations during the four years with the most effectiveness in 1998. Simultaneous
research on entomophthoralean infection of A. pisum and cereal aphids at the same locality
did not confirmed the fungal activity since no N. fresenii infection was recorded within the
aphid populations /ŠTALMACHOVÁ and CAGÁŇ 2000, CAGÁŇ and BARTA 2001/, however
epizootics were observed within colonies of A. fabae /see section 5.3.1/. N. microlophii was
discovered and described for the first time among dispersed colonies of M. carnosum in
Switzerland. The pathogen usually occurred between mid-June and mid-July /KELLER 1991/.
It has also been observed in Poland in the stage of resting spores within a specimen of the
common nettle aphid filled by resting spores of C. obscurus /BAŁAZY 1993/. During our
investigation the pathogen affected the aphid populations every year but not in the year 2002.
It seems to be a common pathogen of the aphid species and it is the most probably
monophagous pathogen so its direct influence on other aphid species within a landscape is
low if any. E. planchoniana is more frequent in dry and moderately humid habitats and
usually does not occur in moist sites /BAŁAZY 1993/. It prefers aphid communities on tall
plants, bushes, and trees /KELLER 1987a/. This may be a reason why the pathogen was so
scarce in the quite humid microhabitat of dense nettle patches at the locality. During our
survey on the Entomophthorales in aphid populations in Slovakia C. obscurus was identified
altogether from 17 different aphid species (see section 5.1.1). The fungus was present within
colonies at low levels besides other pathogens as an occasional species and the disease
development never reached epizootic character. The minor position of C. obscurus was also
observed in M. carnosum colonies. Further two fungi, C. coronatus and Z. phalloides,
reported from literature as pathogens of M. carnosum were not observed at the locality. C.
coronatus is considered to exist mainly as a widespread soil saprophyte, nevertheless the
species is known to cause disease in insects of different orders but also in mammals
/MACLEOD and MÜLLER-KÖGLER 1973, KELLER 1987a/. In regard to Z. phalloides, the aphid-
specific pathogen was originally described from M. carnosum in Poland /BATKO 1966a/ and
now the fungus is known from different species of aphids in Europe and North America
/BAŁAZY 1993/. Although this pathogen was never found on aphids in annual crops but only
in meadows and natural habitats in Switzerland /KELLER 1991/ we did not find it infecting its
type host at the Slovak locality.
The simultaneous occurrence of the three different entomopathogens within the same aphid
population suggests relatively independent development of the mycoses. However, certain
competitive relations must be taken into consideration as the pathogens utilize the same
substrate. A mixed infection, a case when more than one pathogen develops inside aphid’s
body, have also been recorded /e.g. DEAN and WILDING 1971, BAŁAZY 1993/. But possible
interactions (positive or antagonistic) among the pathogens remain still questionable. Anyway
coincident development of more diseases at one site may, on the other hand, enhance
effectiveness of the natural enemies. While for augmentation biological control an isolate with
high virulence should by selected, for conservation biological control a diverge range of
fungal species or isolates of certain species should be encouraged in the environment.
Analysis of the mutual relationship of single elements in the association composed of
entomophthoralean pathogens, aphids on U. dioica, and some injurious aphid species
inhabiting the same environment showed some connections among them. Successful results of
transmission experiments with the pathogens among different aphid species may indicate not
only a possible role of economically unimportant aphid species as alternative hosts for
epizootic development of fungal diseases, but also a role of infection reservoir in ecosystems.
A successful cross-infection between variety of aphids species has been recently documented
for P. neoaphidis, as well /SHAH et al. 2004/. Aphids of different species that occupy different
host plants and do not interact directly can, theoretically, still influence each other’s
population growth rates if they share fungal diseases. In accordance with this hypothesis if
146
one aphid species increases in abundance the infection rate may also increase and
consequently the second aphid species in the vicinity of infection source may suffer higher
mortality. This interspecific interactions could work but under particular circumstances.
Regardless of favourable environmental conditions inevitable for disease initiation and
development, the presence of susceptible aphid species in sufficient numbers and adequate
concentrations of fungal inocula are necessary at the same place and time. Migration of hosts
and effective distribution of infective inocula in time and space is important, as well.
Entomophthoralean fungi possess excellent property of autodissemination by shooting off
conidia /PELL et al. 2001/ nevertheless they are mostly dependent upon abiotic and biotic
agents for their dispersal within greater distances. Of the abiotic agents air currents may play
the most important role / WILDING 1975, HEMMATI 1999 in PELL et al. 2001, HEMMATI et al.
2001b/. As direct biotic agents for inoculum dispersal may serve aphids themselves. Aphids
may spread infection among colonies either inside the same crop or among different crops.
Since M. carnosum is monophagous species its contribution to direct spreading of infection to
adjacent crops is limited. It is obvious that both arthropod enemies and entomopathogenic
fungi co-exist in large aphid populations in the same habitats. There are examples in the
literature when hymenopteran parasitoids or predators acted as passive vectors of
entomophthoralean fungi to host populations during foraging /POPRAWSKI et al. 1992, PELL et
al. 1997a, ROY et al. 2001/, but there are also examples where this did not occur /FURLONG
and PELL 1996, FUENTES-CONTRERAS et al. 1998/ and even predators might contribute to
removal of inocula from the environment by feeding fungus infected aphids /PELL et al.
1997a/. Several parasitoids can be observed in populations of M. carnosum, but polyphagous
aphid parasitoid Aphidius ervi Haliday is the key-species out of the spectrum /STARÝ 1983/.
Wide specificity of the parasitoid enhances its probability of passive disease vector among
plenty of aphid colonies and aphid species. In this case competitive interactions between
aphid-specific parasitoids and fungal abundance should be taken into consideration /POWEL et
al. 1986b/.
As we mentioned above, for effective role of Urtica patches as fungal refugia and resources
of infective inocula for their dispersal to adjacent aphid populations a temporal synchronism
between phenology of the common nettle aphid (a donor of infection) and pestiferous aphids
(recipients of infection) within a certain ecosystem is important. Certain period of time is
assumed to be needed to initiate and establish entomophthoralean infection in the aphid
colonies and produce a sufficient amount of conidia. Therefore the most convenient situation
would be if development of nettle aphid populations were advanced to population
development of injurious aphids. As most pestiferous aphid species in the locality appear at
the beginning of June /CAGÁŇ 1991, ŠTALMACHOVÁ and CAGÁŇ 2000, CAGÁŇ and BARTA
2001/ the circumstance in 2001 when infection of nettle aphid started already in April and
established during May appears to be most favourable.
The hypothesis of weeds as reservoirs for entomopathogenic fungi is supported by
observations of KELLER and SUTER /1980/ or STEENBERG and EILENBERG /1995/, which
suggest that epizootics of aphid pathogens may be initiated from aphid population in weeds.
In this respect, there are also highlighted perennial cultures by some authors. 30% more
aphids were infested by fungi on peas grown in the neighbourhood of perennial Leguminosae
plants that in the peas in the neighbourhood of annual crops. Pea aphid overwinters as eggs,
mainly on perennial Leguminosae plants, so that in the spring aphid development likewise the
development and presence of their natural reducers could take place earlier /MAJCHROVICZ
2001/. KELLER and SUTER /1980/ found out that on annual plants in agrocenoses, in which
much alfalfa is cultivated, aphid populations were restricted by numerous natural enemies,
including mycosis. Though, this seems to be site and year-depended. During our intensive
monitoring of aphids and their pathogens at Dolná Malanta only few aphids were observed on
147
alfalfa plots and no infection was present. Similar observations were made at the locality in
the previous study on seasonal dynamics and entomophthoralean infection of pea aphid
/CAGÁŇ and BARTA 2001/. During that experiment few P. neoaphidis cadavers were only
found at the end of June in 1999 when the aphid population density was decreasing. The
problem of epizootic development among several aphid species is more complex. It is obvious
that not all the aphidophagous fungi may spread equally among different aphid species. Our
results of transmission experiments show that two aphid species were not infected by
pathogens collected in nature. Reasons given for the failure may be different. A range of host
specificity of particular pathogen also limits disease distribution within ecosystems. For
instance, N. microlophii is probably monophagous species therefore its function, from
viewpoint of interspecific interaction in landscape, seems to be irrelevant. Even virulence of
strains of oligophagous pathogens may differ to various aphid species. Two types of C.
obscurus strains with different virulence against A. pisum and S. avenae was documented
/PAPIEROK and WILDING 1981/. And what is more, populations and biotypes of the same
aphid species may differ significantly in their susceptibility to particular strain of pathogen.
Two different biotypes of A. pisum were significantly different in their susceptibility to P.
neoaphidis isolates /MILNER 1982/.
The biological diversity of not only aphid-specific entomophthoralean fungi and their
biotypes but also aphids and their biotypes within agroecosystems is unquestionably rather
great. Other studies of interactions among the organisms at three trophic levels (host plant,
aphids, pathogens) are necessary to clarify factors governing development of epizootics
within environment in the presence of different aphid species and different pathogens. New
knowledge would make easier manipulation of floral components of agroecosystems to
provide improved and sustainable insect pest management.
148
6. CONCLUSIONS
6. CONCLUSIONS
15 entomophthoralean fungal species out of three families were identified from 70 aphid
species collected randomly in various localities in Slovakia. Out of the 15 fungal species 9 are
the first records from Slovakia (P. nouryi, Z. radicans, Z. aphidis, Z. phalloides, Z.
occidentalis, C. thromboides, N. microlophii, N. cinarae, and N. turbinata) and one is the first
record from central Europe (E. erinacea). The fungi could be observed in the aphid
populations from April to November with most records in May and October. Four fungal
species, P. neoaphidis, E. planchoniana, N. fresenii, and C. obscurus, were most frequently
identified from aphids during the study. The fungal diseases were recorded from aphids at
non-production sites as well as at fields. Many of the records of entomophthoralean fungi
were from important pestiferous aphids, e.g. A. pisum, M. persicae, R. padi, R. maidis, M.
dirhodum, S. avenae, D. noxia, and A. fabae.
In Austria 7 fungal species were identified from aphid hosts. All of the species are the first
record from the country.
Four fungal species, Pandora neoaphidis, Conidiobolus obscurus, Conidiobolus
thromboides, and Zoophthora radicans, were successfully isolated and cultivated on artificial
media. Overall 20 isolates of Slovak provenience were obtained from dead animals.
Pandora uroleuconii was described as a new species. The new taxon was described from
its type host Uroleucon aeneum and it was compared and contrasted with similar species of
the genus.
A key to identify aphid-parasiting Entomophthorales is included in this study. The key,
based primarily on morphological features as well as on host preferences and world
distribution of pathogens, takes account of all the entomophthoralean species, which have
been presented in literature as aphid pathogens.
---
Certain biological characteristics of 15 Pandora neoaphidis isolates were tested in their
variability. Phenotypic characteristics evaluated in the trials revealed a significant
intraspecific variability among the Slovak isolates. The variability was also demonstrated by
isolates obtained from the same host colonies during epizootics.
The isolates showed great heterogeneity in the conidial size. In vitro produced primary
conidia were significantly greater that those collected from killed aphids.
Estimates of lethal concentrations of conidia (LC 50 ) of P. neoaphidis isolates ranged from
24 to 214 conidia per mm2 for the pea aphid, with average virulence of 138.35 conidia per
mm2. The virulence was significantly different among isolates.
Higher proportion of primary spores germinated at saturated atmosphere than at 75%
relative humidity. Conidia exposed to 75% humidity did not preserve viability and did not
germinate when they were subsequently exposed to saturated atmosphere. Character of
surface, on which conidia are deposited, can influence a type of conidial germination. A
germ-tube formation prevailed over a secondary conidium formation on a glass surface,
whereas secondary conidia were almost exclusively produced on an agar surface. Relationship
between conidial germination on glass and virulence of the isolates showed a strong negative
relationship indicating that the level of conidial germination on glass could be reflective of
germination of conidia in vivo on host cuticle.
P. neoaphidis isolates showed a good growth on the culture medium with a daily rate of
radial growth of 35.03 - 116.67 mm2. There was a significant difference in radial growth rate
among the isolates.
Average biomass production of P. neoaphidis isolates in liquid medium ranged between
1.63 to 8.72 g.l-1. The biomass production was statistically different among the isolates.
---
149
6. CONCLUSIONS
A pattern of seasonal population abundance of aphids monitored at the locality of Dolná

Malanta can be describe to show a typical growth curve with two peaks observed at the end of
May and in October. Prevalence of entomophthoralean infection reflected the pattern of live
aphids, though the peaks were about a week delayed.
Altogether, 6 parasitic fungi from the order Entomophthorales were identified on 18 aphid
species regularly monitored at Dolná Malanta. They were P. neoaphidis, Z. aphidis, E.
planchoniana, N. fresenii, N. microlophii, and C. obscurus. Further two fungal species E.
erinacea and Z. radicans were collected randomly from additional two aphid species. P.
neoaphidis was the most prevalent species and a source of epizootics in colonies of A. pisum
and M. dirhodum. N. fresenii was secondary to P. neoaphidis in occurrence, commonly
infecting several aphid species and causing epizootics in A. fabae colonies. Despite several
epizootics were observed in the aphid populations at the locality, the fungal infections
normally appeared after aphids had become damaging on the crops. The group of fungi only
sporadically attacked spring generations of fundatrices.
Using descriptive analysis of spatial distribution we observed that prevalence of
entomophthoralean infection grew with aphid infestation of sample units, e.g. plants with
higher aphid infestation were more often bearing fungus-killed aphids. As a rule, the
pathogens started to be more active when host populations were decreasing in abundance
indicating that high abundance of host is probably not as important for epizootic development
as physiological state of the hosts. Disease spread out slower within population of more
sedentary and aggregated A. fabae than of more mobile and randomly dispersed M. dirhodum
and A. pisum.
Negative relationship between aggregation of aphids and number of plants infested with
aphids was detected for all aphid species and aphid mortality was in general positively
dependent upon the aphid aggregation. Taylor’s power law provided a highly significant
relationship between variance and mean density per sample unit. This analysis fitted well for
the both live and fungus-killed aphids and estimated slopes indicated aggregated spatial
distribution for live and fungus-killed aphids. The degree of aggregation represented by
Taylor’s slope coefficients appeared to be similar between live and fungus-killed aphids.
Relationship between the proportion of sample units bearing aphid cadavers (P 1 ) and the
mean numbers of cadavers per sample unit provided the possibility to estimate of fungal
prevalence in the crop by counting the number of sample units bearing fungus-killed aphids,
since the modeled P 1 values correlated well to the values of P 1 observed in the crops. Sample
size curves for fixed levels of precision were also developed for four aphid-pathogen-habitat
systems. Generally, more samples were needed for more aggregated aphid species.
Stinging nettle patches can be considered as fugal parasite reservoirs in agricultural
landscape. Aphids regularly infesting the stinging nettle were annually found suffering from
entomophthoralean diseases. Transmission of entomophthoralean disease between the nettle
aphid and various aphid species co-occurring in the landscape was successful, suggesting a
possible role of non-crop vegetation as a source of the infection for aphids injurious in
agriculture.
150
7. CONTRIBUTION OF THE WORK FOR PRACTICAL USE
7.CONTRIBUTION OF THE WORK FOR PRACTICAL USE

Development of insecticide resistance in aphid populations and increasing public concern
over deleterious effects of pesticide use on human health lead to investigations into alternative
aphid control strategies. Recently, the development of entomophthoralean fungi as aphid
biocontrol agents has received increasing interest as part of integrated control strategies.
Preliminary investigation on natural occurrence of the group of organisms among aphid
populations colonising crop or non-crop vegetation is necessary step for development of
prospective biocontrol agent in successive step.
In Slovakia a great variety of entomophthoralean species have been detected within aphid
populations in agroecosystems demonstrating a convoluted interactions between hosts and
their fungal enemies. These results also give a good background for further study of the
natural enemies with intention of their use in strategies of biological control. At one regularly
visited locality 8 parasitic fungi were identified from different aphids. Most of them were
active in pestiferous aphid colonies and provided epizootics in the host colonies. Out of the
fungal complex associated with aphids in the country, four entomophthoralean species, P.
neoaphidis, E. planchoniana, N. fresenii, and C. obscurus, were observed annually in colonies
of pestiferous aphids and can be considered as important mortality factors of aphids in crops.
All these species are aphid-specific and also show ability to provide a natural control of new
invasive aphid species, which recently have become naturalized under conditions of Slovakia.
Unfortunately as it can be seen from our results a simply rely on the natural enemies would
not give an appropriate control of aphids in growing cultures, since the fungi as a rule start to
be effective after aphids has reached a damage level. One or combination of more
programmes of biological control strategies thus should be applied in order to control the
pests. We successfully evaluated a link between pestiferous and harmless aphid species
occupying vegetation in agroecosystems through entomophthoraleans. Harmless aphid species
can share their entomophthoralean enemies with pest species inhabiting the same ecological
niches in agroecosystem. This implies a possible application of conservation biological
control in the pest management strategies in order to support non-pest aphids and their
enemies thus building a reservoir of enemies in the system. However, it would be worthwhile
to study in more detail how elements of landscape and cropping system enter into the trophic
interactions of “alternative host – parasite – pest host”, as well as how a man-manipulated
conditions may enhance entomophthoraleans in the landscape. Augmentation programmes are
auspicious with some species easily cultivated and produced in vitro. However, besides
troubles with formulation, viability of inocula and biopesticide compatibility with
agrochemicals or cropping system, one should consider whether releasing of single virulent
isolate as an element of biopreparation is satisfactory for success. Since consortia of strains
are probably active in host population, even in the course of epizootics, as some of our results
indicate.
151
8. SUMMARY
8. SUMMARY
Study on occurrence of parasitic fungi from the order Entomophthorales in populations of
various aphids was carried out in Slovakia during 1999-2002. A great diversity of fungal
species was observed during the survey. The fungi could be observed in aphid populations
from April to November and altogether 15 entomophthoralean species were identified from
70 aphid species. 9 fungal species, P. nouryi, Z. radicans, Z. aphidis, Z. phalloides, Z.
occidentalis, C. thromboides, N. microlophii, N. cinarae, and N. turbinata, are considered the
first record from Slovakia and E. erinacea is the first record from central Europe. Seasonal
distribution, host spectrum and prevalence of each species are individually discussed and
compared with results of other authorities in the world. Four fungal species, P. neoaphidis, E.
planchoniana, N. fresenii, and C. obscurus, were most frequently identified from aphids
during the study. Many of the records of entomophthoralean fungi were from important
pestiferous aphids, e.g. A. pisum, M. persicae, R. padi, R. maidis, M. dirhodum, S. avenae, D.
noxia, and A. fabae. Epizootics were observed in some aphid colonies. During the study a new
species was described. A fungus attacking dense colonies of Uroleucon aeneum on Carduus
nutans was described as a new species of the genus Pandora.
Similar survey on aphid parasiting fungi was carried out in Austria during 2000-2001 and 7
fungal species were identified from aphid cadavers. All the seven entomophthoralean species
found in Austria are considered the first record from the country. The four most common
species in Slovakia were also frequent in Austria.
A key to identify aphid-parasiting Entomophthorales is presented in this study. The key
includes all entomophthoralean species, which have been presented in literature as aphid
pathogens.
Four fungal species, Pandora neoaphidis, Conidiobolus obscurus, Conidiobolus
thromboides, and Zoophthora radicans, were successfully isolated and cultivated on artificial
media. 15 isolates of P. neoaphidis, 3 isolates of C. thromboides, and 1 isolate of C. obscurus
and Z. radicans were obtained from aphid cadavers. The P. neoaphidis isolates were included
into bioassay to compare certain phenotypic characteristics of species. A significant
heterogeneity was observed among the isolates tested even though some isolates had the same
origin. Average virulence (LC 50 ) of the P. neoaphidis isolates for the pea aphid was 138.35
conidia per mm2.
Patterns of seasonal population abundance of 18 pest or non-pest aphids and their fungal
enemies were monitored at Dolná Malanta during 2001-2002. In general a growth curve of
population abundance showed two peaks and prevalence of entomophthoralean infection
reflected the pattern of live aphids, though the peaks were about a week delayed. P.
neoaphidis and N. fresenii were the only fungal species with epizootic capacity at the locality,
however the disease did not prevent the crops from damage by aphids. A variety of
endogenous and exogenous factors, which may act together, are involved in aphid
susceptibility to fungal diseases and thus may influence a disease development within aphid
populations. Epizootic patterns of two entomophthoralean fungi developing in aphid
populations with different degree of spatial aggregation were compared, with intention to
evaluate whether a spatial heterogeneity in aphid density may influence a disease
development. Disease spread out slower within population of more sedentary and aggregated
A. fabae than of more mobile and randomly dispersed M. dirhodum and A. pisum. Taylor’s
power law revealed an aggregated spatial distribution for live and fungus-killed aphids and
spatial distribution of fungus-killed aphids was not statistically different from that of live
aphids. A positive relationship between aphid mortality and aphid aggregation of live aphids
was mostly detected.
152
8. SUMMARY
Relationship between the proportion of plants bearing aphid cadavers and the mean
numbers of cadavers per plants revealed that disease in more aggregated aphid species need
more time to spread out within population than in less aggregated species. Sample size curves
for fixed levels of precision were also developed for different aphid-pathogen-habitat systems.
Generally, more samples were needed for more aggregated aphid species.
Stinging nettle patches were evaluated as pathogen reservoirs in agricultural landscape.
Aphids regularly infesting the stinging nettle were often found suffering from
entomophthoralean diseases. Transmission of entomophthoralean disease between the nettle
aphid and various aphid species co-occurring in the landscape was successful, suggesting a
possible role of non-crop vegetation as a source of the infection for aphids injurious in
agriculture.
153
9. SÚHRN
9. SÚHRN
Počas rokov 1999-2002 sme sledovali výskyt entomopatogénnych húb z radu
Entomophthorales v populáciách rôznych druhov vošiek na Slovensku. Výsledky prieskumu
poukazujú na druhovú rozmanitosť parazitických húb v kolóniách vošiek. Rovnako sme zistili
druhovú pestrosť vošiek, na ktorých sa hubové ochorenie prejavilo. Spolu sme na Slovensku
identifikovali 15 druhov entomopatogénnych húb na 70 druhoch vošiek. Parazitované vošky
bolo možné objaviť od apríla až do novembra. 9 druhov, P. nouryi, Z. radicans, Z. aphidis, Z.
phalloides, Z. occidentalis, C. thromboides, N. microlophii, N. cinarae a N. turbinata, sú
uvedené na Slovensku po prvýkrát a druh E. erinacea je po prvýkrát zistený v oblasti strednej
Európy. V práci bližšie charakterizujeme hostiteľské spektrum jednotlivých druhov patogénov
na Slovensku, ich sezónne rozšírenie a prevalencia ochorenia v kolóniách vošiek. Výsledky
porovnávame s pozorovaniami iných autorov vo svete. V podmienkach Slovenska sme zistili
4 druhy entomopatogénnych húb, P. neoaphidis, E. planchoniana, N. fresenii a C. obscurus,
ktoré sa počas prieskumu objavovali najčastejšie v kolóniách vošiek. Tieto patogény sa
najväčšou mierou podieľali na vzniku hubových ochorení poľnohospodársky významných
druhov vošiek, napr. A. pisum, M. persicae, R. padi, R. maidis, M. dirhodum, S. avenae, D.
noxia a A. fabae. Bolo zaznamenaných aj niekoľko epizoótií v kolóniách vošiek. V práci sme
popísali nový druh entomopatogénnej huby z radu Entomophthorales, ktorý sme zistili na
voške Uroleucon aeneum žijúcej na bodliaku, Carduus nutans. Taxón bol popísaný ako nový
druh pre rod Pandora.
V rokoch 2000-2001 sme uskutočnili rovnaký prieskum entomopatogénnych húb vošiek
v Rakúsku. Zistili sme 7 druhov patogénnych húb v populáciách rôznych druhov vošiek.
Všetky druhy sú pre Rakúsko evidované ako nový nález. 4 najviac zastúpené druhy na
Slovensku boli najčastejšie aj v podmienkach Rakúska.
V dizertačnej práci sme zostavili aj jednoduchý kľúč, ktorý by mal pomôcť pri identifikácii
všetkých húb z radu Entomophthorales uvedených v literatúre ako patogény vošiek.
4 druhy patogénov, Pandora neoaphidis, Conidiobolus obscurus, Conidiobolus
thromboides a Zoophthora radicans, sme úspešne izolovali na umelých živných pôdach
a ďalej kultivovali v podmienkach in vitro. Celkovo sme získali 15 izolátov druhu P.
neoaphidis, 3 izoláty patogéna C. thromboides a po 1 izoláte druhov Z. radicans a C.
obscurus. Izoláty druhu P. neoaphidis sme použili v laboratórnych testoch s cieľom posúdiť
niektoré fenotypické znaky druhu a ich variabilitu medzi izolátmi. Testy preukázali významnú
heterogenitu vo viacerých sledovaných znakoch. Variabilita bola preukazná aj medzi izolátmi,
ktoré mali spoločný pôvod, t.j. boli izolované z rovnakej populácie vošiek pri epizoótii
ochorenia. Priemerná hodnota letálnej koncentrácie spór (LC 50 ) izolátov druhu P. neoaphidis
bola 138.35 konídií na mm2 pre vošku hrachovú.
Na lokalite Dolná Malanta sme v rokoch 2001-2002 sledovali sezónnu dynamiku 18
druhov vošiek a prevalenciu hubového ochorenia v kolóniách týchto vošiek. Rastová krivka
abundancie populácie v priebehu sezóny mala dva vrcholy, jarný a jesenný. Vo všeobecnosti
prevalencia hubového ochorenia v populáciách týchto vošiek odrážala dynamiku populácie
vošiek, no vrcholy abundancie mŕtvych vošiek v kolóniách boli oneskorené asi o týždeň. P.
neoaphidis a N. fresenii boli jediné druhy, ktoré v uvedenej lokalite vyvolali epizoótiu
v populáciách niektorých vošiek. Existuje veľké množstvo faktorov, ktoré vo vzájomnej
súčinnosti vstupujú do systému patogén – hostiteľ a ovplyvňujú jeden alebo oba prvky tohto
systému, a tak môžu zasahovať do vývoja ochorení v populáciách hmyzu. V práci sme
sledovali priebeh epizoótií dvoch patogénnych húb v kolóniách rôznych druhov vošiek, pre
ktoré bol typický rozličným stupeň agregácie, s cieľom posúdiť možný vplyv priestorového
rozmiestnenia hostiteľa na priebeh ochorenia. Zistili sme, že ochorenie sa šírilo pomalšie
v populáciách vošky makovej, pre ktorú je typické rozmiestnenie v zhlukoch v prostredí, než
154
9. SÚHRN
v populáciách pohyblivejšej vošky trávovej a hrachovej s vyšším stupňom náhodného

rozmiestnenia. Taylorova analýza poukázala na zhlukovité priestorové rozmiestnenie u živých
aj mŕtvych vošiek v sledovaných porastoch, pričom miera disperzie nebola štatisticky
preukazne rozdielna medzi oboma sledovanými skupinami. Zistili sme pozitívnu koreláciu
medzi mortalitou vošiek a mierou agregácie živých vošiek na hostiteľských rastlinách.
Sledovali sme závislosť medzi podielom rastlín s mŕtvymi voškami a priemerným počtom
mŕtvych vošiek na rastlinu. Pre vošky so náhodnejším priestorovým rozmiestnením je
pravdepodobne potrebný kratší čas na rozšírenie ochorenia v populácii než pre vošky s viac
zhlukovitým rozmiestnením. Pre viaceré systémy voška – patogén – hostiteľská rastlina boli
stanovené krivky pre výpočty potrebného množstva hodnotených rastlín pre stanovenie
prevalencie ochorenia v populáciách pri rôznych stupňoch presnosti odhadu. Vo všeobecnosti
možno povedať, že pre druhy vošiek so zhlukovitou disperziou je potrebné vyhodnotiť vyšší
počet rastlín pri rovnakom stupni presnosti odhadu.
Hodnotili sme nesúvislé porasty žihľavy v lokalite Dolná Malanta s cieľom posúdiť ich
význam ako zdroj patogénov vošiek v agroekosystéme. Vošky žijúce na žihľave boli
pravidelne infikované patogénnymi hubami z radu Entomophthorales. Uskutočnili sme preto
umelý prenos infekcie medzi voškami na žihľave a voškami žijúcimi v systéme na
poľnohospodárskych plodinách. Úspešný prenos ochorenia vytvára priestor na diskusiu
o možnej úlohe neplodinovej vegetácie v agroekosystéme ako zdroja prirodzených
nepriateľov vošiek škodiacich na pestovaných plodinách.
155
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188
11. Appendix
11. APPENDIX
189
11. Appendix
Table 25. List of isolates of entomophthoralean fungi cultivated in vitro
Isolates: Source for isolation: Culture Locality, Date

(No. of isolate, species) (Host aphid, host plant) medium: of isolation:
1. Pandora neoaphidis Myzus persicae (Sulzer), Nicotiana SDAYEM* Veľký Kýr,
(Rem. et Henn.) Humber tabaci Linné 7.11. 2000
2. Pandora neoaphidis Myzus persicae (Sulzer), Nicotiana SDAYEM Komjatice,
3. Pandora neoaphidis Myzus persicae (Sulzer), Nicotiana SDAYEM Veľký Kýr,
4. Pandora neoaphidis Myzus persicae (Sulzer), Brassica SDAYEM Komjatice,
(Rem. et Henn.) Humber oleracea Linné 5.12. 2000
5. Pandora neoaphidis Microlophium carnosum (Buckton), SDAYEM Dolná Malanta,
(Rem. et Henn.) Humber Urtica dioica Linné 18.4. 2001
8. Pandora neoaphidis Sitobion avenae (Fabricius), SDAYEM Komjatice,
(Rem. et Henn.) Humber Triticum aestivum Linné, 21.5. 2001
9. Pandora neoaphidis Sitobion avenae (Fabricius), SDAYEM Komjatice,
(Rem. et Henn.) Humber Triticum aestivum Linné 21.5. 2001
10. Conidiobolus Therioaphis trifolii (Monell), SDAYEM Zbehy,
thromboides Drechsler Medicago sativa Linné 10.9. 2001
13. Pandora neoaphidis Uroleucon aeneum (Hille Ris SDAYEM Komjatice,
(Rem. et Henn.) Humber Lambers), Carduus nutans Linné 13.6. 2002
14. Pandora neoaphidis Uroleucon aeneum (Hille Ris SDAYEM Veľký Cetín,
(Rem. et Henn.) Humber Lambers), Carduus nutans Linné 22.6. 2002
15. Conidiobolus obscurus Myzus persicae (Sulzer), Persica SDAYEM Komjatice,
(Hall et Dunn) Rem. et vulgaris Linné 4.10. 2002
Keller
16. Pandora neoaphidis Microlophium carnosum (Buckton), SDAYEM Námestovo,
17. Pandora neoaphidis Microlophium carnosum (Buckton), SDAYEM Sliač,
18. Pandora neoaphidis Rhopalosiphum padi (Linné), Zea SDAYEM Vlkas,
(Rem. et Henn.) Humber mays Linné 15.10. 2002
19. Pandora neoaphidis Rhopalosiphum padi (Linné), Zea SDAYEM Komjatice,
(Rem. et Henn.) Humber mays Linné 21. 10. 2002
20. Zoophthora radicans Brevicoryne brassicae (Linné), SDAYEM Dolná Malanta,
(Brefeld) Batko Brassica napus Linné 9.9. 2002
____________________________________________________________
*SDAYEM - Sabouraud dextrose agar, yeast extract, milk, and egg yolks
11. Appendix
Table 26. Biometrics of important fungal structures used in the identification

Slovak material Austrian material
Pandora neoaphidis (Remaudière et Hennebert) Humber
Primary conidia: L*: 21.46-28.12 (17.82-35.64) µm L: 21.32-31.48 (17.82-35.64) µm
D*: 10.77-14.53 (9.90-17.82) µm D: 11.01-16.93 (9.90-21.78) µm
L/D*: 1.62-2.23 L/D: 1.65-2.48
(50 series*) (96 series)
Hyphal bodies: D: 12.46-12.82 (9.90-15.84) µm
―
(4 series)
Nuclei: 6.00-8.95 (3.96-10.89) µm 5.25-9.98 (3.71-11.88)µm
(20 series) (25 series)
Secondary conidia L: 16.75-22.18 (13.86-25.74) µm L: 16.04-24.57 (13.21-26.04) µm
(type Ia): D: 9.23-13.51 (8.91-15.84) µm D: 10.15-16.04 (9.90-19.90) µm
L/D: 1.44-1.92 L/D: 1.30-2.02
(type Ib): D: 14.97-15.84 (13.86-16.83) µm D: 14.27-18.51 (11.88-20.79) µm
L/D: 1.32-1.35 L/D: 1.29-1.43
Rhizoids: D: 15.84 (7.92-23.76) µm
―
(1 series)
Pseudocystidia: L: 306.90 (158.40-495.00) µm
D: 12.95 (7.92-17.82) µm
(1 series)
Pandora uroleuconii Barta et Cagáň
Hyphal bodies: L: 120.6 (66.8-200.5) µm (2 series)
D: 9.28-10.08 (7.92-12.87) µm ―
(4 series)
Conidiophores: 10.18-11.33 (9.90-12.87) µm
―
(2 series)
Primary conidia: L: 24.71-33.03 (21.78-41.58) µm
D: 11.57 -17.19 (9.90-22.77) µm
―
L/D: 1.79-2.25
(10 series)
Nuclei: 5.46-7.09 (3.96-8.91) µm (8 series) ―
Secondary conidia L: 18.14-19.21 (15.84-27.72) µm
(type Ia): D: 9.31-9.62 (7.92-11.88) µm
―
L/D: 1.93-2.01
(4 series)
Secondary conidia L: 17.27-18.45 (11.88-27.72)
(type Ib): D: 9.58-10.18 (7.92-11.88)
―
L/D: 1.70-1.93
(4 series)
Pseudocystidia: L: 176.6 (138.6 – 207.9) µm
(1 series)
D: 15.46 (11.88 – 19.80) µm
―
(13 objects) diameter at base
9.5 (7.92 – 11.88) µm
(1series) diameter in the middle
11. Appendix
5.99 (5.18 – 6.35) µm

(1 series) diameter at apex
Pandora nouryi (Remaudière et Hennebert) Humber
D: 10.65-10.85 (8.91-13.86) µm
―
L/D: 1.55-1.58
(4 series)
Resting spores: 25.62-27.24 (23.76-31.68) µm
―
(4 series)
Primary conidia: 15.56-16.32 (13.86-17.82) µm ―
Resting spores: 28.59-33.62 (23.76-35.64) µm 30.69-32.12 (23.76-37.62) µm
D: 7.26-8.00 (5.94-8.91) µm
―
L/D: 2.38-2.52
(4 series)
Nuclei: 4.37-4.75 (2.97-5.94) µm (4 series) ―
Capillary 37.18-37.30 (23.76-51.48) µm
―
conidiophores: (2 series)
(type II): D: 5.09-5.61 (3.96-6.93) µm ―
L/D: 3.37-3.55
Zoophthora aphidis (Hoffman in Fresenius) Batko
Primary conidia: L: 28.99-30.10 (23.76-35.64) µm L: 28.59-30.06 (23.76-35.64) µm
D: 12.87-15.13 (10.89-17.82) µm D: 12.27-15.00 (10.89-17.82) µm
L/D: 1.99-2.31 L/D: 2.00-2.33
Nuclei: 6.85-7.33 (3.96-9.90) µm (4 series) 6.25-7.11 (3.96-9.90) µm
(4 series)
(type II): D: 12.67-13.42 (9.90-15.84) µm D: 12.30-13.55 (9.90-15.84) µm
L/D: 2.19-2.23 L/D: 2.18-2.34
Capillary 67.64-72.47 (37.62-99.00) µm 65.12-75.69 (37.62-99.00) µm
conidiophores: (4 series) (4 series)
―
(4 series)
Zoophthora occidentalis (Thaxter) Batko
D: 12.47-14.18 (10.89-17.82) µm
―
L/D: 2.43-2.63
(4 series)
(type I): D: 11.52-11.76 (9.90-13.86) µm
―
L/D: 1.98-2.06
(4 series)
11. Appendix

Primary conidia: L: 33.42-34.08 (26.30-44,71) µm
D: 11.41-11.84 (10.52-15.78) µm
―
L/D: 2.84-2.99
(4 series)
Entomophthora planchoniana Cornu
D: 13.44-18.33 (11.88-21.78) µm D: 12.83-19.33 (11.20-21.78) µm
L/D: 1.09-1.28 L/D: 1.13-1.35
Nuclei: 3.29-3.83 (2.38-5.94) µm 3.05-4.16 (2.38-5.94) µm
Secondary conidia: L: 13.25-15.05 (11.88-15.84) µm
D: 10.44-12.35 (9.90-13.86) µm
―
L/D: 1.22-1.27
(4 series)
Neozygites fresenii (Nowakowski) Remaudière et Keller
D: 13.50-17.36 (11.88-19.80) µm D: 13.82-17.59 (7.92-19.80) µm
L/D: 1.12-1.48 L/D: 1.12-1.35
(19series) (39 series)
(type II): D: 10.38-17.15 (9.90-19.80) µm D: 11.40-18.25 (8.91-19.80) µm
L/D: 1.41-2.48 L/D: 1.30-2.65
Capillary 20.46-66.29 (15.84-97.02) µm 20.36-66.13 (7.92-102.96) µm
conidiophores: (14 series) (39 series)
Resting spores: L: 41.35-43.02 (36.00-46.80) µm
D: 21.75-22.20 (19.80-25.20) µm
―
L/D: 1.90-1.94
(4 series)
D: 29.59-29.65 (25.20-34.20) µm
―
L/D: 1.20-1.22
(4 series)
(type II): D: 11.10-13.95 (10.80-18.00) µm
―
L/D: 2.14-2.32
(4 series)
Capillary 95.95-108.14 (50.40-270.00) µm
―
Neozygites cinarae Keller
D: 14.31-15.06 (11.88-19.80) µm D: 14.85-15.25 (11.88-19.80) µm
L/D: 1.69-1.70 L/D: 1.65-1.68
―
(type II): D: 14.05-15.98 (13.45-16.80) µm
11. Appendix
L/D: 2.10-2.24
(2 series)
Resting spores: L: 32.17-33.58 (27.07-38.50) µm L: 32.26-33.66 (27.72-37.62) µm
D: 22.18-23.03 (20.14-27.88) µm D: 22.33-23.36 (20.79-26.73) µm
L/D: 1.45-1.46 L/D: 1.39-1.45
Resting spores: L: 37.94-38.41 (31.68-45.54) µm
D: 22.97-23.13 (19.80-25.74) µm
―
L/D: 1.64-1.66
(4 series)
Capillary 72.77 (57.42-89.10) µm
Germ conidia: L: 33,34-33.62 (29.70-37.62) µm
D: 11.92-12.32 (9.90-13.86) µm
―
L/D: 2.73-2.80
(2 series)
Conidiobolus obscurus (Hall et Dunn) Remaudière et Keller
Primary conidia: L: 31.05-42.53 (25.74-55.44) µm L: 35.28-37.95 (29.70-44.84)
D: 25.98-36.75 (21.78-49.50) µm D: 29.52-32.23 (25.74-37.62)
L/D: 1.09-1.24 L/D: 1.14-1.23
D: 24.62-27.72 (23.76-31.68) µm
―
L/D: 1.22-1.23
(4 series)
―
(6 series)
Conidiobolus thromboides Drechsler
D: 18.61-20.99 (16.83-23.76) µm
―
L/D: 1.28-1.32
(6 series)
D: 16.04-17.94 (13.86-19.80) µm
―
L/D: 1.24-1.27
(6 series)
___________________________
*L – length
D – diameter
L/D – length/diameter ratio
1 series = measurements of 25 objects
― data not measured
11. Appendix
Tables 27-41. Complete lists of entomophthoralean species, their host aphids collected in
Slovakia during 1999-2002 including localities and dates of collecting
Table 27. Pandora neoaphidis (Remaudière et Hennebert) Humber

Aphid species Host plant Locality Date
1. Acyrthosiphon pisum (Harris) Pisum sativum Linné 25.5. 1999
10.6. 2000
Dolná Malant
12.6. 2001
21.5. 2002
Komjatice 25.5. 1999
Uľany nad
12.7. 2001
Žitavou
Rastislavice 26.6. 2001
Žemberovce 2.7. 2002
Medicago sativa Linné 30.5. 1999
Dolná Malanta 17.6. 2000
21.5. 2002
15.6. 2000
Komjatice
19.5. 2001
Šurany 20.6. 2001
Veľké Bielice 5.9. 2001
Mužľa 22.10. 2001
Vicia sp. Rabča 18.6. 2002
2. Aphis acetosae subg. acetosae Rumex acetosa Linné
Bobrovník 2.7. 2002
Linné
3. Aphis fabae Scopoli unknown Strážne 6.7. 2001
Euonymus europaea
Linné
Heracleum
Staré Hory 2.7. 2002
sphondylium Linné
Faba vulgaris Moench. Dolná Malanta 12.6. 2002
Beta vulgaris Linné Dolná Malanta 12.6. 2002
Aphis fabae subg. Cirsium arvense Linné Vyšné Hágy 6.7. 2001
cirsiiacanthoidis Scopoli
Valča 17.6. 2002
Habovka 17.6. 2002
Rabča 18.6. 2002
Zliechov 1.7. 2002
Námestovo 1.7. 2002
Aphis fabae subg. mordwilkoi Arctium lappa Linné Huncovce 6.7. 2001
Börner et Janisch
Veľký Cetín 30.5. 2002
11. Appendix
Rabča 18.6. 2002

4. Aphis frangulae gossipii Chrysanthemum sp.
Glover
5. Aphis idaei van der Goot Rubus idaeus Linné Habovka 1.7. 2002
6. Aphis pomi De Geer Malus domestica
Borkh.
7. Aphis sambuci Linné Sambucus nigra Linné 30.10. 2001
Dolná Malanta
21.5. 2002
Oravský
17.6. 2002
Podzámok
8. Aphis urticata Gmelin Urtica dioica Linné 31.5. 2001
Dolná Malanta
9.5. 2002
Streda nad
10.5. 2002
Bodrogom
9. Brachycaudus cardui (Linné) Helianthus annuus Rastislavice 14.6. 2001
Linné
Bajč 22.6. 2001
Mudroňovo 22.6. 2001
Carduus nutans Linné Komjatice 3.6. 2002
10. Brachycaudus helichrysi Erigeron canadensis Kamenica nad
20.5. 2002
(Kaltenbach) Linné Hronom
Chľaba 20.5. 2002
11. Brachycaudus lychnidis Melandrium album
(Linné) (Mill.)
12. Brachycorynella asparagi Asparagus officinalis 25.9. 2000
Komjatice
(Mordvilko) Linné 30.9. 2001
13. Brevicoryne brassicae (Linné) Brassica napus Linné Dolná Malanta 21.6. 1999
2.7. 1999
Brassica oleracea Trávnica 9.10. 2001
Linné
Komjatice 1.6. 2002
14. Callipterinella calliptera Betula pendula Ehrh.
Nitra 22.9. 1999
(Hartig)
15. Capitophorus horni Börner Cirsium arvense Linné Komjatice 23.6. 1999
16. Cavariella pastinacae (Linné) Heracleum
mantegazzianum Slaská 23.5. 2001
Sommier et Lévier
17. Cavariella theobaldi (Gillette Heracleum
and Bragg) mantegazzianum Slaská 23.5. 2001
Sommier et Lévier
18. Cinara pilicornis (Hartig) Picea abies Karst. Oravská
17.6. 2002
Polhora
19. Cryptomyzus gaelopsidis Ribes rubrum Linné 25.9. 1999
Komjatice
(Kaltenbach) 14.10. 1999
20. Cryptomyzus korschelti Börner Ribes rubrum Linné 25.9. 1999
Komjatice
14.10. 1999
11. Appendix
21. Cryptomyzus ribis (Linné) Ribes rubrum Linné Komjatice 15.4. 2001
22. Diuraphis noxia (Mordvilko) Hordeum vulgare Dubno 5.7. 2001
Linné
Plášťovce 6.7. 2001
7.5. 2001
Dolná Malanta
26.6. 2002
23. Dysaphis sp. Rumex obtusifolius
Linné
24. Eukalipterus tiliae (Linné) Tilia cordata Mill. Nitra 26.9. 1999
25. Hyalopteroides humilis Dactylis glomerata
Habovka 17.6. 2002
Walker Linné
26. Hyalopterus pruni (Geoffroy) Iris sp. Komjatice 16.7. 1999
27. Hyperomyzus lactucae (Linné) Sonchus arvensis Linné Dolná Malanta 17.6. 1999
28.6. 1999
Komjatice 1.6. 2002
Lactuca sativa Linné Komjatice 13.10. 2000
28. Macrosiphoniella artemisiae Artemisia vulgaris
(Boyer de Fonscolombe) Linné
29. Macrosiphum funestum Rubus idaeus Linné
Harmanec 10.8. 1999
(Macchiati)
30. Macrosiphum rosae (Linné) Rosa canina Linné Kamenica nad
20.5. 2002
Hronom
Oravská
17.6. 2002
Polhora
Rabča 18.6. 2002
Belá 1.7. 2002
Dipsacus silvester Dolná Malanta 4.6. 2002
Hunds.
Valča 17.6. 2002
Rubus idaeus Linné Rabča 18.6. 2002
Rubus fruticosus Linné Kunerad 1.7. 2002
31. Megoura viciae Buckton Lathyrus sp. Habovka 17.6. 2002
Rabča 18.6. 2002
32. Metopolophium dirhodum Rosa canina Linné Nitra 30.9. 1999
(Walker)
Zea mays Linné Kamenica nad
10.9. 1999
Hronom
Sliač 18.6. 2002
11. Appendix
Belá 1.7. 2002

Triticum aestivum 2.5. 2001
Dolná Malanta
Linné 4.6. 2002
Hordeum vulgare Dolná Malanta 4.6. 2002
Linné
Devičie 18.6. 2002
33. Metopolophium festucae Dactylis glomerata Kamenica nad
26.4. 2001
(Theobald) Linné Hronom
34. Microlophium carnosum Urtica dioica Linné 8.6. 1999
(Bukton) Dolná Malanta 3.4. 2001
30.4. 2001
9.5. 2002
Černík 25.6. 1999
Kamenica nad 26.4. 2001
Hronom 20.5. 2002
Pozba 20.5. 2002
Dolný Kubín 17.6. 2002
Habovka 17.6. 2002
Oravská
17.6. 2002
Polhora
Rabča 18.6. 2002
Komjatná 18.6. 2002
Korytnica 18.6. 2002
Motyčky 18.6. 2002
Sliač 18.6. 2002
Kunerad 1.7. 2002
35. Myzus ascalonicus Doncaster Tripleurospermum
inodorum (Linné)
36. Myzus cerasi (Fabricius) Cerasus avium (Linné) 21.5. 2001
Dolná Malanta
Moench. 1.11. 2002
37. Myzus persicae (Sulzer) Solanum tuberosum
Linné
Nicotiana tabacum 12.9. 1999
Linné 27.9. 1999
Veľký Kýr
9.9. 2000
4.10. 2002
7.11. 2000
Komjatice
17.11. 2000
Kamenica nad
22.10. 2001
Hronom
Bešeňov 22.10. 2001
Demandice 22.10. 2001
11. Appendix
21. 10.
Raphanus sativus Linné Komjatice 1999
7. 11. 1999
Lactuca sativa Linné Komjatice 13.10. 2000
Orobanche ramosa
Bešeňov 22.10. 2001
Linné
Brassica napus Linné Komjatice 5.12. 2000
Pastovce 22.10. 2001
Bešeňov 22.10. 2001
Humulus lupulus Linné Dolná Malanta 21.5. 2002
Persica vulgaris Linné Komjatice 20.10. 2002
38. Myzus s. str. sp. Malva sp. Belá 1.7. 2002
39. Periphyllus testudinaceus Acer campestre Linné
(Fernie)
40. Phyllaphis fagi (Linné) Fagus silvatica Linné Komjatice 19.5. 2001
41. Rhopalosiphum maidis (Fitch) Hordeum vulgare 16.10. 2001
Dolná Malanta
Linné 19.6. 2002
42. Rhopalosiphum padi (Linné) Zea mays Linné Dolná Malanta 12.6. 2001
Vlkas 9.10. 2001
Gbelce 22.10. 2001
Bajč 22.10. 2001
Hordeum vulgare
Pastovce 22.10. 2001
Linné
Triticum aestivum Kamenica nad
22.10. 2001
Linné Hronom
Capsella bursa-pastoris Bátorove
22.10. 2001
(Linné) Med. Kosihy
Padus avium Mill. Komjatice 22.4. 2000
10.10. 2001
Dolná Malanta
17.10. 2002
Alopecurus pratensis
Rabča 18.6. 2002
Linné
43. Sitobion avenae (Fabricius) Poa pratensis Linné Bíňa 9.5. 20001
Belá 1.7. 2002
Veľká Lehota 9.5. 2001
Linné
Hordeum vulgare Malé Kosihy 22.10. 2001
Linné
Devičie 18.6. 2002
11. Appendix
Triticum aestivum Komjatice 21.5. 2001

Linné 7.5. 2001
Dolná Malanta
12.6. 2002
Kamenica nad
9.5. 2001
Hronom
Zea mays Linné Dolná Malanta 4.6. 2001
44. Sitobion fragariae (Walker) Rubus idaeus Linné Belá 1.7. 2002
45. Uroleucon aeneum (Hille Ris Carduus nutans Linné 23.6. 1999
Lambers) Komjatice 13.10. 2001
19.5. 2002
Ivanka pri Nitre 16.5. 2002
Nitra 30.5. 2002
Malý Cetín 30.5. 2002
Valča 17.6. 2002
Habovka 17.6. 2002
Zliechov 1.7. 2002
Fačkov 1.7. 2002
Kunerad 1.7. 2002
Korytnica 2.7. 2002
Kostoľany pod
11.7. 2002
Tribečom
Sonchus oleraceus
Linné
Crepis setosa Hall. Kláštor pod
17.6. 2002
Znievom
Dolný Kubín 17.6. 2002
Rabča 18.6. 2002
Ružomberok 18.6. 2002
Belá 1.7. 2002
Zázrivá 1.7. 2002
Oravská
2.7. 2002
Polhora
Cichorium intybus Sliač 18.6. 2002
Linné
Devičie 18.6. 2002
46. Uroleucon campanulae Chrysanthemum sp. 20.10. 2001
Komjatice
(Kaltenbach) 4.11. 2001
47. Uroleucon erigeronensis unknown Habovka 17.6. 2002
11. Appendix
(Thomas) Filipendula ulmaria Oravská

17.6. 2002
(Linné) Max. Polhora
unknown Rabča 18.6. 2002
48. Uroleucon tanaceti (Linné) Tanacetum vulgare
Zázrivá 1.7. 2002
Linné
11. Appendix
Table 28. Entomophthora planchoniana Cornu

1. Aphis fabae Scopoli Euonymus europaea 10.10. 2001
Dolná Malanta
Linné 9.5. 2002
2. Aphis pomi De Geer Malus domestica Borkh. Dolná Malanta 14.5. 2001
3. Aphis sambuci Linné Sambucus nigra Linné Komjatice 13.10.1999
Kamenica nad
7.6. 2001
Hronom
4. Aphis symphyti Schrank Symphytum officinale
Oravská Polhora 17.6. 2002
Linné
5. Brachycaudus cardui (Linné) Carduus nutans Linné Ružový Dvor 24.6. 2001
6. Brachycaudus helichrysi Erigeron canadensis Kamenica nad
20.5. 2002
(Kaltenbach) Linné Hronom
Chľaba 20.5. 2002
7. Capitophorus carduinus Carduus nutans Linné
Mužľa 13.6. 2001
(Walker)
8. Capitophorus horni Börner Cirsium arvense Linné Komjatice 23.6. 1999
Sommier et Lévier
Salix sp. Dolná Malanta 7.5. 2001
Sommier et Lévier
11. Chaetosiphon tetrarhodum Rosa canina Linné 30.6. 2001
Dolná Malanta
(Walker) 10.10. 2001
12. Cryptomyzus ribis (Linné) Ribes rubrum Linné Komjatice 8.4. 2001
13. Diuraphis noxia (Mordvilko) Hordeum vulgare Linné Dolná Malanta 23.6. 2001
14. Drepanosiphum platanoidis 16.9. 1999
Acer sp. Nitra
(Schrank) 8.10. 1999
15. Dysaphis plantaginea (Passerini) Malus domestica Borkh. Dolná Malanta 14.5. 2001
16. Hyadaphis foeniculi (Passerini) Conium maculatum
Selice 3.6. 2001
Linné
17. Hyalopterus pruni (Goeffroy) Prunus spinosa Linné Dolná Malanta 21.5. 2001
Kamenica nad
7.6. 2001
Hronom
Phragmites australis 4.6. 2001
Dolná Malanta
(Cav.) Trin. 19.6. 2002
18. Macrosiphum euphorbiae Kamenica nad
Rosa canina Linné 20.5. 2001
(Thomas) Hronom
19. Macrosiphum rosae (Linné) Rosa canina Linné Kamenica nad
20.5. 2001
Hronom
11. Appendix
20. Metopolophium dirhodum Zea mays Linné Kamenica nad

10.9. 1999
(Walker) Hronom
Rosa canina Linné Dolná Malanta 10.10. 2001
21. Microlophium carnosum Urtica dioica Linné
(Buckton)
22. Myzus ascalonicus Doncaster Tripleurospermum
inodorum (Linné)
23. Myzus cerasi (Fabricius) Cerasus avium (Linné)
Moench
24. Myzus persicae (Sulzer) Persica vulgaris Mill. 10.10. 1999
Komjatice
20.10. 2002
Nicotiana tabacum Bešeňov 22.10. 2001
Linné Veľký Kýr 4.10. 2002
Humulus lupulus Linné Nitra 24.5. 2002
25. Rhopalosiphum padi (Linné) Zea mays Linné Kamenica nad 10.9. 1999
Hronom 13.6. 2001
Hordeum vulgare Linné Mojzesovo 9.10. 2001
Padus avium Mill. 27.4. 2001
14.5. 2001
Dolná Malanta
10.10. 2001
15.4. 2002
26. Sitobion avenae (Fabricius) Alopecurus pratensis
Linné
Triticum aestivum Linné Dolná Malanta 12.6. 2002
27. Sitobion fragariae (Walker) Rubus idaeus Linné Streda nad
10.5. 2002
Bodrogom
11. Appendix
Table 29. Neozygites fresenii (Nowakowski) Remaudière et Keller

1. Aphis acetosae subg. acetosae Rumex acetosa Linné Malá nad
9.5. 2001
Linné Hronom
Ondrochov 12.5. 2001
Harmanec 30.5. 2001
Kamenica nad
20.5. 2002
Hronom
2. Aphis craccae (Linné) Vicia sp. Dolné Krškany 9.7. 1999
3. Aphis fabae Scopoli Euonymus europaea 30.9. 1999
Linné 2.5. 2001
Dolná Malanta
16.10. 2001
27.9. 2002
Chenopodium album
Linné
Faba vulgaris Moench. 2.5. 2001
Dolná Malanta
21.5. 2001
12.6. 2002
Beta vulgaris Linné 4.6. 2001
Dolná Malanta
12.6. 2002
Heracleum spondylium
Belá 1.7. 2002
Linné
Aphis fabae subg. Cirsium arvense (Linné) Harmanec 30.5. 2001
Cirsiiacanthoidis Scopoli
Zliechov 1.7. 2002
Aphis fabae subg. Mordwilkoi Arctium lappa Linné Komjatice 17.5. 2001
Börner et Janisch
Kamenica nad
7.6. 2001
Hronom
4. Aphis nasturtii Kaltenbach Capsicum annuum 8.7. 1999
Komjatice
Linné 2.6. 2002
Rhamnus cathartica
Linné
5. Aphis pomi De Geer Malus domestica Borkh. Komjatice 22.5. 2002
6. Aphis rumicis Linné Rumex obtusifolius Lukáčovce 31.8. 2001
Linné
Ondrochov 4.9. 2001
Kamenica nad
20.5. 2001
Hronom
11. Appendix
Capitophorus elaeagni Elaeagnus angustifolia Nitra 21.9. 1999
8. (del Guercio) Linné
Vlkas 9.10. 2001
Hippophae rhamnoides
Nitra 26.9. 1999
Linné
Sommier et Lévier
Sommier et Lévier
11. Cryptomyzus gaelopsidis Ribes rubrum Linné
(Kaltenbach)
12. Euceraphis betulae Koch Betula pendula Ehrh. Nitra 4.10. 1999
13. Hyadaphis foeniculi (Passerini) Heracleum spondylium
Komjatice 7.9. 1999
Linné
14. Hyalopterus pruni (Goeffroy) Phragmites australis Dolný Jatov 6.8. 1999
(Cav.) Trin. Spišské
6.7. 2001
Podhradie
Prunus domestica Linné Dolná Malanta 31.5. 2001
15. Macrosiphum funestum Rubus idaeus Linné
Harmanec 10.8. 1999
(Macchiati)
16. Macrosiphum rosae (Linné) Rosa canina Linné Nitra 30.9. 1999
17. Metopolophium dirhodum Hordeum vulgare Linné Dolná Malanta 19.6. 2002
(Walker) Zea mays Linné Dolná Malanta 19.6. 2002
18. Microlophium carnosum Urtica dioica Linné 8.7. 1999
Dolná Malanta
(Bukton) 4.6. 2001
Kamenica nad
7.6. 2001
Hronom
19. Myzus cerasi (Fabricius) Cerasus avium (Linné) Nitra 4. 10. 1999
Moench
Komjatice 5. 10. 1999
20. Myzus persicae (Sulzer) Persica vulgaris Mill. Komjatice 17.9. 1999
Daucus carota Linné Komjatice 28.10. 1999
21. Periphyllus testudinaceus Acer campestre Linné
(Fernie)
22. Rhopalosiphum padi (Linné) Padus avium Mill. Komjatice 20.9. 1999
10.10. 2001
Dolná Malanta
20.9. 2002
Vlkas 9.10. 2001
Capsella bursa-pastoris
Trávnica 9.10. 2001
(Linné) Med.
11. Appendix
Triticum aestivum Linné Komjatice 8.10. 2001

23. Uroleucon aeneum (Hille Ris Carduus nutans Linné Mužľa 13.6. 2001
Lambers)
Zázrivá 1.7. 2002
Linné
11. Appendix
Table 30. Conidiobolus obscurus (Hall et Dunn) Remaudière et Keller

25.6. 2000
Dolná Malanta
30.6. 2001
4.6. 2002
2. Aphis fabae Scopoli Beta vulgaris Linné Dolná Malanta 12.6. 2001
Faba vulgaris Moench. Dolná Malanta 12.6. 2002
Aphis fabae subg. Cirsium arvense Linné
Habovka 17.6. 2002
4. Aphis sambuci Linné Sambucus nigra Linné Oravský
17.6. 2002
Podzámok
5. Brevicoryne brassicae (Linné) Brassica oleracea Linné Komjatice 28.10. 1999
mantegazzianum Vyšné Hágy 6.7. 2001
Sommier et Lévier
and Bragg) mantegazzianum Vyšné Hágy 6.7. 2001
Sommier et Lévier
8. Macrosiphoniella artemisiae Artemisia vulgaris
(Boyer de Fonscolombe) Linné
9. Macrosiphum rosae (Linné) Dipsacus silvester
Valča 17.6. 2002
Huds.
10. Megoura viciae Buckton Lathyrus sp. Habovka 17.6. 2002
Oravská polhora 2.7. 2002
11. Metopolophium dirhodum Hordeum vulgare Linné
(Walker)
12. Microlophium carnosum Urtica dioica Linné Pozba 20.5. 2002
(Bukton)
13. Myzus persicae Sulzer Persica vulgaris Linné Komjatice 20.10. 2002
14. Rhopalosiphum padi (Linné) Triticum aestivum Linné Dolná Malanta 3.10. 2001
8.10. 2001
Komjatice
3.11. 2002
Padus avium Mill. 30.10. 2001
Dolná Malanta
27.9. 2002
Rabča 18.6. 2002
Linné
Hordeum vulgare Linné Dolná Malanta 19.6. 2002
15. Rhopalosiphum maidis (Fitch) Triticum aestivum Linné Komjatice 3.11. 2002
Hordeum vulgare Linné Dolná Malanta 19.6. 2002
11. Appendix
16. Uroleucon aeneum (Hille Ris Carduus nutans Linné 2.10. 2001
Lambers) Komjatice 7.10. 2001
13.10. 2001
13.6. 2002
Černík
23.6. 2002
Sonchus oleraceus 3.10. 2001
Komjatice
Linné 5.10. 2001
12.10. 2001
Cichorium intybus
Nitra 8.10. 2001
Linné
Cirsium arvense (Linné)
Nitra 12.5. 2002
Scop.
Crepis setosa Hall. Dolný Kubín 17.6. 2002
17. Uroleucon campanulae Chrysanthemum sp.
(Kaltenbach)
Table 31. Pandora uroleuconii Barta et Cagáň

1. Uroleucon aeneum (Hille Ris Carduus nutans 23.6. 1999
Komjatice
Lambers) Linné 19.5. 2002
Kamenica nad
23.5. 2001
Hronom
Rastislavice 12.6. 2001
Mužľa 13.6. 2001
3.6. 2001
Ružový Dvor
2.7. 2001
Dubno 5.7. 2001
Spišské Podhradie 6.7. 2001
Malý Cetín 30.5. 2002
Valča 17.6. 2002
Habovka 17.6. 2002
Černík 23.6. 2002
Nitra 30.6. 2002
Zliechov 1.7. 2002
Korytnica 2.7. 2002
Jatov 6.7. 2002
Kláštor pod Znievom 17.6. 2002
Kostoľany pod
11.7. 2002
Tribečom
11. Appendix
Table 32. Zoophthora radicans (Brefeld) Batko

2. Chaitophorus leucomelas Koch Populus nigra Linné Selice 3.6. 2001
3. Myzus persicae (Sulzer) Persica vulgaris Mill. Komjatice 17.9. 1999
Table 33. Zoophthora aphidis (Hoffman in Fresenius) Remaudière et Hennebert

1. Anoecia corni (Fabricius) 6.11. 2001
Cornus sanguinea Linné Dolná Malanta
20.9. 2002
3. Myzus cerasi (Fabricius) Cerasus avium (Linné)
Moench
4. Rhopalosiphum padi (Linné) Padus avium Mill. 15.10. 2001
Dolná Malanta
25.10. 2002
Table 34. Neozygites turbinata (Kenneth) Remaudière et Keller

1. Pterocomma salicis (Linné) Salix sp. Komjatice 21.7. 2001
2. Tuberolachnus salignus
Salix sp. Nitra 3.10. 2001
(Gmelin)
Table 35. Neozygites microlophii Keller

1. Microlophium carnosum 8.7. 1999
(Bukton) Urtica dioica Linné Dolná Malanta 2.5. 2001
4.6. 2002
Table 36. Neozygites cinarae Keller

1. Cinara pilicornis (Hartig) Slaská 1.8. 2001
Picea abies Karst.
Čavoj 1.7. 2002
11. Appendix
Table 37. Conidiobolus thromboides Drechsler

1. Therioaphis trifolii (Monell) Zbehy 31.8. 2001
Medicago sativa Linné
Veľké Bielice 5.9. 2001
Table 38. Pandora nouryi (Remaudière et Hennebert) Humber

1. Aphis acetosae subg. acetosae
Rumex acetosa Linné Komjatice 13.10. 2002
Linné
Table 39. Erynia erinacea (Ben-Ze’ev et Kenneth) Remaudière et Hennebert

1. Aphis umbrella (Börner) Malva neglecta Wallr. Dolná Malanta 17.9. 2002
Table 40. Zoophthora phalloides Batko

1. Cinara pilicornis (Hartig) Picea abies Karst. Čavoj 1.7. 2002
Table 41. Zoophthora occidentalis (Thaxter) Batko

Zázrivá 1.7. 2002
Linné
11. Appendix
Tables 42-48. Complete lists of entomophthoralean species, their host aphids collected in
Austria during 2000-2001 including localities and dates of collecting.
Table 42. Pandora neoaphidis (Remaudière et Hennebert) Humber

1. Acyrthosiphon neerladicum
Euphorbia esula Linné Wien 21.9. 2001
Hille Ris Lambers
2. Acyrthosiphon
Malva sp. Wimpassing 8.11. 2001
pelargonii Kaltenbach
Fuchsenbigl
25.9. 2001
Eidletzberg 24.9. 2001
Wimpassing 8.11. 2001
4. Capsella bursa-pastoris
Aphis craccivora Koch Groβ-Enzersdorf 22.9. 2001
(Linné) Med.
5. Euonymus europaea
Aphis fabae subg. fabae Scopoli Wien 16.10. 2000
Linné
Aphis fabae subg.
Cirsium arvense Linné Wien 27.6.2001
6. Aphis nasturtii Kaltenbach Cucurbita pepo Linné Groβwilfersdorf 12.7. 2001
7. Aphis sambuci Linné Sambucus nigra Linné Fuchsenbigl 10.11. 2001
8. Helianthus annuus
Brachycaudus cardui (Linné) Wien 27.6.2001
Linné
9. Brachycorynella asparagi Asparagus officinalis 17.10. 2000
Fuchsenbigl
(Mordvilko) Linné 25.9. 2001
10. Brevicoryne brassicae (Linné) Brassica napus Linné Breitstetten 17.10. 2000
11. Capitophorus elaeagni Elaeagnus angustifolia Wien 17.10. 2000
(del Guercio) Linné Fuchsenbigl 10.11. 2001
12. Capitophorus horni subg. horni
Cirsium arvense Linné Wien 27.6.2001
Börner
13. Cavariella theobaldi (Gillette et Lycopersicum
Fuchsenbigl 28.6. 2001
Bragg) esculentum Mill.
14. Cavariella pastinacae (Linné) Lycopersicum
esculentum Mill.
15. Diuraphis noxia (Mordvilko) Hordeum vulgare Linné Fuchsenbigl 28.6. 2001
16. Dysaphis crataegi (Kaltenbach) Crataegus sp. Wien 10.10. 2000
17. Hyperomyzus lactucae (Linné) Sonchus arvensis Linné Groβwilfersdorf 12.7. 2001
18. Macrosiphum funestum
Rubus fructicosus Linné Gosau 11.7. 2001
(Macchiati)
19. Metopolophium dirhodum Rosa canina Linné Wien 16.10. 2000
(Walker) Wimpassing 8.11. 2001
20. Microlophium carnosum
Urtica dioica Linné Neuwaldegg 28.6. 2001
(Buckton)
21. Myzus persicae (Sulzer) Prunus persica Mill. Fuchsenbigl 9.10. 2000
11. Appendix
Daucus carota Linné Schafflerhof 11.10. 2000

Lycopersicum
esculentum Mill.
Brassica napus Linné Woppendorf 9.11. 2001
17.10. 2000
Fuchsenbigl
10.11. 2001
Raasdorf 11.10. 2000
Sinapis alba Linné
Woppendorf 9.11. 2001
22. Phorodon cannabis Passerini Cannabis sativa Linné Eidletzberg 24.9. 2001
23. Hordeum vulgare Linné Groβ-Enzersdorf 22.9. 2001
Rhopalosiphum maidis (Fitch) Triticum aestivum
Linné
24. Rhopalosiphum padi (Linné) Zea mays Linné Fuchsenbigl 9.10. 2000
Schafflerhof 11.10. 2000
Wien 20.9. 2001
Neu-oberhausen 22.9. 2001
Hordeum vulgare Linné Süβenbrunn 23.9. 2001
Triticum aestivum Breitstetten 9.10. 2000
Linné
Capsella-bursa pastoris
(Linné) Med.
25. Sitobion avenae (Fabricius) Triticum aestivum
Breitstetten 9.10. 2000
Linné
Zea mays Linné Schafflerhof 11.10. 2000
26. Uroleucon aeneum (Hille Ris Centaurea scabiosa
Lambers) Linné
11. Appendix
Table 43. Entomophthora planchoniana Cornu

1. Aphis fabae subg. fabae Scopoli Euonymus europaeus
Linné
2. Aphis pomi De Geer Wimpassing 8.11. 2001
Malus domestica Borkh.
3. Aphis sambuci Linné Sambucus nigra Linné Fuchsenbigl 28.6. 2001
Wien 28.6. 2001
4. Eucallipterus tiliae (Linné) Wien 28.6. 2001
Tilia cordata Mill.
Groβwilfersdorf 12.7. 2001
5. Capitophorus elaeagni Elaeagnus angustifolia Fuchsenbigl 10.11. 2001
(del Guercio) Linné
Wien 11.11. 2001
6. Cavariella pastinacae (Linné) Pastinaca sativa Linné Neuwaldegg 10.7. 2001
7. Cavariella theobaldi (Gillette et
Salix caprea Linné Gosau 11.7. 2001
Bragg)
8. Metopolophium dirhodum
Rosa canina Linné Wien 16.10. 2000
(Walker)
9. Rhopalosiphum padi (Linné) Zea mays Linné Schafflerhof 11.10. 2000
10. Sitobion avenae (Fabricius) Zea mays Linné Schafflerhof 11.10. 2000
Table 44. Conidiobolus obscurus (Hall et Dunn) Remaudière et Keller

1. Acyrthosiphon pisum (Harris) Pisum sativum Linné Fuchsenbigl 28.6. 2001
2. Aphis umbrella (Börner) Malva neglecta Wallr. Wien 21.9. 2001
3. Rhopalosiphum padi (Linné) Triticum aestivum Linné Woppendorf 9.11. 2001
Table 45. Zoophthora aphidis (Hoffman in Fresenius) Batko

1. Anoecia corni (Fabricius) Cornus sanguinea Linné Wien 8.11. 2001

Table 46. Neozygites cinarae Keller

1. Cinara pilicornis (Hartig) Picea abies Karst. Gosau 11.7. 2001
11. Appendix
Table 47. Neozygites fresenii (Nowakowski) Remaudière et Keller

1. Aphis acetosae subg. Acetosae Rumex sp. Groβwilfersdorf 12.7. 2001
Linné Eidletzberg 24. 9. 2001
2. Aphis fabae subg. Fabae Euonymus europaea
Wien 16.10. 2000
Scopoli Linné
Faba vulgaris Moench Fuchsenbig 28.6. 2001
Chenopodium album
Groβwilfersdorf 12.7. 2001
Linné
Aphis fabae subg. mordwilkoi
Arctium lappa Linné Neuwaldegg 28.6. 2001
Börner et Janisch
Aphis fabae subg.
Cirsium arvense Linné Gosau 11.7. 2001
Cirsiiacanthoidis Scopoli
4. Aphis nasturtii Kaltenbach Rhamnus cathartica Wien 16.10. 2000
Linné Fuchsenbigl 10. 11. 2001
5. Brachycaudus cardui (Linné) Carduus sp. Gosau 11.7. 2001
6. Capitophorus elaeagni Elaeagnus angustifolia Wien 17.10. 2000
(del Guercio) Linné Fuchsenbigl 10.11. 2001
7. Cryptomyzus ribis (Linné) Ribes rubrum Linné Wien 6.10. 2000
8. Hyalopterus pruni (Geoffroy) Phragmites australis
Wien 13.7. 2001
(Cav.) Trin.
9. Rhopalosiphum maidis (Fitch) Hordeum vulgare Linné Groβ-Enzersdorf 22.9. 2001
10. Rhopalosiphum padi (Linné) Padus avium Linné. Wien 6.10. 2000
Hordeum vulgare Linné Süβenbrunn 23.9. 2001
Zea mays Linné Fuchsenbigl 25.9. 2001
Table 48. Erynia erinacea (Ben-Ze’ev et Kenneth) Remaudière et Hennebert

1. Aphis umbrella (Börner) Malva neglecta Wallr. Wien 21.9. 2001
11. Appendix
Table 49. Numbers of living and fungus-killed aphids recorded from 45 twigs of Malus domestica Borkh. at Dolná Malanta in 2001
Aphis pomi De Geer Dysaphis plantaginea (Passerini)

2001 Living aphids Dead aphids Living aphids Dead aphids
Date Alate Apteral ∑ Alate Apteral ∑ Alate Apteral ∑ Alate Apteral ∑
30.3. 0 0 0 0 0 0 0 89 89 0 0 0
12.4. 0 0 0 0 0 0 0 78 78 0 0 0
19.4. 0 14 14 0 0 0 0 104 104 0 0 0
27.4. 0 21 21 0 0 0 0 69 69 0 0 0
2.5. 0 134 134 0 0 0 0 235 235 0 0 0
7.5. 11 61 72 0 0 0 2 493 495 0 0 0
14.5. 0 0 0 0 0 0 0 346 346 0 2EP 2
21.5. 3 29 32 0 0 0 1 272 273 0 0 0
31.5. 0 0 0 0 0 0 7 589 596 0 0 0
4.6. 2 6 8 0 0 0 40 758 798 3EP 43EP 46
12.6. 1 8 9 1EP 0 1 0 45 45 0 2EP 2
23.6. 1 20 21 0 0 0 3 96 99 0 0 0
30.6. 0 26 26 0 0 0 0 21 21 0 0 0
7.7. 0 8 8 0 0 0 1 43 44 0 0 0
15.7. 0 3 3 0 0 0 0 0 0 0 0 0
3.10. 0 0 0 0 0 0 0 0 0 0 0 0
16.10. 3 0 3 0 0 0 0 0 0 0 0 0
30.10. 2 0 2 0 0 0 0 0 0 0 0 0
6.11. 0 4 4 0 0 0 0 0 0 0 0 0
14.11. 0 0 0 0 0 0 1 0 1 0 0 0
EP – aphids killed with Entomophthora planchoniana

11. Appendix
Table 50. Numbers of living and fungus-killed aphids recorded from 45 twigs of Malus domestica Borkh. at Dolná Malanta in 2002
Aphis pomi De Geer Dysaphis plantaginea (Passerini)

2002 Living aphids Dead aphids Living aphids Dead aphids
Date Alate Apteral ∑ Alate Apteral ∑ Alate Apteral ∑ Alate Apteral ∑
28.3. 0 0 0 0 0 0 0 0 0 0 0 0
2.4. 0 0 0 0 0 0 0 2 2 0 0 0
9.4. 0 0 0 0 0 0 0 0 0 0 0 0
16.4. 0 0 0 0 0 0 0 0 0 0 0 0
2.5. 0 0 0 0 0 0 0 0 0 0 0 0
9.5. 0 0 0 0 0 0 0 0 0 0 0 0
14.5. 0 0 0 0 0 0 0 0 0 0 0 0
21.5. 0 0 0 0 0 0 0 0 0 0 0 0
31.5. 0 0 0 0 0 0 0 0 0 0 0 0
4.6. 0 0 0 0 0 0 0 0 0 0 0 0
10.9. 1 0 1 0 0 0 0 0 0 0 0 0
17.10. 26 7 33 1PN+1ZA 0 2 0 0 0 0 0 0
25.10. 57 105 162 2PN+6NF 0 8 0 0 0 0 0 0
1.11. 3 30 33 2CO 0 2 0 5 5 0 0 0
PN – aphids killed with Pandora neoaphidis

ZA – aphids killed with Zoophthora aphidis
NF – aphids killed with Neozygites fresenii
CO – aphids killed with Conidiobolus obscurus
11. Appendix
Table 51. Numbers of living and fungus-killed aphids recorded from 45 twigs of Sambucus nigra Linné at Dolná Malanta in 2001 and 2002
Aphis sambuci Linné Aphis sambuci Linné

2001 Living aphids Dead aphids 2002 Living aphids Dead aphids
Date Alate Apteral ∑ Alate Apteral ∑ Date Alate Apteral ∑ Alate Apteral ∑
30.3. 0 2 2 0 0 0 2.4. 0 2 2 0 0 0
3.4. 0 0 0 0 0 0 9.4. 0 0 0 0 0 0
12.4. 0 2 2 0 0 0 16.4. 0 11 11 0 0 0
19.4. 0 3 3 0 0 0 25.4. 0 385 385 0 0 0
27.4. 0 652 652 0 0 0 2.5. 0 3055 3055 0 0 0
7.5. 0 900 900 0 0 0 9.5. 0 9090 9090 0 0 0
14.5. 0 2600 2600 0 0 0 14.5. 66 8139 8205 0 0 0
31.5. 13 2447 2460 0 0 0 21.5. 196 4433 4629 0 1PN 1
8.6. 138 1727 1865 2EP 3EP 5 28.5. 30 633 663 0 0 0
12.6. 43 1326 1369 0 0 0 4.6. 0 110 110 0 0 0
30.6. 0 10 10 0 0 0 12.6. 0 65 65 0 0 0
7.7. 1 10 11 0 0 0 19.6. 0 90 90 0 0 0
15.7. 0 2 2 0 0 0 1.9. 1 0 1 0 0 0
3.10. 2 4 6 0 0 0 10.9. 2 22 24 0 0 0
16.10. 20 89 109 1EP 1EP 2 27.9. 2 11 13 0 0 0
30.10. 1 7 8 4EP 1PN 0 5 2.10. 2 20 22 0 0 0
25.10. 29 0 29 0 0 0
11. Appendix
Table 52. Numbers of living and fungus-killed aphids recorded from 45 twigs of Rosa canina Linné at Dolná Malanta in 2001
Macrosiphum rosae (Linné) Chaetosiphon tetrarhodum (Walker) Metopolophium dirhodum (Walker)

2001 Living aphids Dead aphids Living aphids Dead aphids Living aphids Dead aphids
Date Alate Apteral ∑ Alate Apteral ∑ Alate Apteral ∑ Alate Apteral ∑ Alate Apteral ∑ Alate Apteral ∑
30.3. 0 3 3 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
1.4. 0 67 67 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
12.4. 0 128 128 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
19.4. 0 292 292 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
27.4. 0 743 743 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
2.5. 35 381 436 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
7.5. 31 909 940 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
14.5. 42 394 436 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
21.5. 44 151 195 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
31.5. 40 284 324 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
4.6. 12 215 227 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
12.6. 6 366 372 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
23.6. 0 240 240 0 0 0 0 98 98 0 3EP 3 0 0 0 0 0 0
30.6. 0 12 12 0 0 0 0 185 185 0 4EP 4 0 0 0 0 0 0
15.7. 0 0 0 0 0 0 0 16 16 0 0 0 0 0 0 0 0 0
22.7. 0 0 0 0 0 0 0 55 55 0 0 0 0 0 0 0 0 0
27.7. 0 0 0 0 0 0 0 116 116 0 0 0 0 0 0 0 0 0
3.8. 0 0 0 0 0 0 0 51 51 0 0 0 0 0 0 0 0 0
10.8. 0 0 0 0 0 0 0 110 110 0 0 0 0 0 0 0 0 0
17.8. 0 0 0 0 0 0 0 370 370 0 0 0 0 0 0 0 0 0
24.8. 0 0 0 0 0 0 0 690 690 0 0 0 0 0 0 0 0 0
30.8. 0 0 0 0 0 0 0 107 107 0 0 0 0 0 0 0 0 0
11.9. 0 0 0 0 0 0 0 108 108 0 0 0 0 0 0 0 0 0
19.9. 0 0 0 0 0 0 0 104 104 0 0 0 0 0 0 0 0 0
3.10. 0 0 0 0 0 0 0 5 5 0 0 0 2 21 23 1EP 1EP 2
10.10 0 0 0 0 0 0 0 3 3 3EP 0 3 30 211 241 8EP 5EP 13
16.10 0 0 0 0 0 0 0 0 0 0 0 0 9 122 131 4EP 1EP 5
30.10 0 0 0 0 0 0 0 0 0 0 0 0 1 56 57 1EP 1EP 2
11. Appendix
Table 53. Numbers of living and fungus-killed aphids recorded from 45 twigs of Rosa canina Linné at Dolná Malanta in 2002
Macrosiphum rosae (Linné) Chaetosiphon tetrarhodum (Walker) Metopolophium dirhodum (Walker)

2.4. 0 0 0 0 0 0 0 0 0 0 0 0 0 87 87 0 0 0
9.4. 0 0 0 0 0 0 0 0 0 0 0 0 0 234 234 0 0 0
16.4. 0 0 0 0 0 0 0 0 0 0 0 0 0 402 402 0 0 0
25.4. 0 0 0 0 0 0 0 0 0 0 0 0 6 283 289 0 0 0
2.5. 0 0 0 0 0 0 0 0 0 0 0 0 6 276 282 0 0 0
9.5. 14 294 308 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
14.5. 26 1320 1346 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
21.5. 51 2597 2648 1EP 0 1 0 0 0 0 0 0 0 0 0 0 0 0
28.5. 58 1525 1583 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
12.6. 10 45 55 0 0 0 1 15 16 0 0 0 0 0 0 0 0 0
19.6. 11 77 88 0 0 0 1 115 116 0 0 0 0 0 0 0 0 0
26.6. 0 2 2 0 0 0 0 177 177 0 0 0 0 0 0 0 0 0
3.7. 0 0 0 0 0 0 0 145 145 0 0 0 0 0 0 0 0 0
8.7. 0 0 0 0 0 0 0 172 172 0 0 0 0 0 0 0 0 0
15.7. 0 6 6 0 0 0 0 142 142 0 0 0 0 0 0 0 0 0
29.7. 0 0 0 0 0 0 0 298 298 0 0 0 0 0 0 0 0 0
5.8. 0 0 0 0 0 0 0 145 145 0 0 0 0 0 0 0 0 0
16.8. 0 0 0 0 0 0 0 3 3 0 0 0 0 0 0 0 0 0
21.8. 0 1 1 0 0 0 0 26 26 0 0 0 0 0 0 0 0 0
26.8. 0 0 0 0 0 0 0 45 45 0 0 0 0 0 0 0 0 0
1.9. 0 0 0 0 0 0 2 591 593 0 0 0 0 0 0 0 0 0
10.9. 1 0 1 0 0 0 5 444 449 0 0 0 0 0 0 0 0 0
20.9. 2 0 2 0 0 0 1 229 230 0 0 0 0 0 0 0 0 0
2.10. 2 0 2 0 0 0 0 35 35 0 0 0 0 0 0 0 0 0
17.10. 0 0 0 0 0 0 0 0 0 0 0 0 110 943 1053 4PN 0 4
25.10. 0 2 2 0 0 0 0 0 0 0 0 0 101 681 782 0 0 0
11. Appendix
Table 54. Numbers of living and fungus-killed aphids of species Hyalopterus pruni (Geoffroy) recorded from 45 twigs of Prunus domestica Linné or 45
leaves of Phragmites australis (Cav.) Trin. at Dolná Malanta in 2001
Prunus domestica Linné Phragmites australis (Cav.) Trin.

1.4. 0 71 71 0 0 0 1.4. 0 0 0 0 0 0
12.4. 0 18 18 0 0 0 12.4. 0 0 0 0 0 0
19.4. 0 21 21 0 0 0 19.4. 0 0 0 0 0 0
27.4. 0 81 81 0 0 0 27.4. 0 0 0 0 0 0
2.5. 0 203 203 0 0 0 2.5. 0 0 0 0 0 0
7.5. 0 1205 1205 0 0 0 7.5. 0 0 0 0 0 0
14.5. 0 7160 7160 0 0 0 14.5. 0 0 0 0 0 0
21.5. 21 29240 29261 0 0 0 21.5. 25 250 275 0 0 0
31.5. 0 12175 12175 1NF 0 1 31.5. 113 1010 1123 0 0 0
4.6. 682 4081 4763 0 0 0 4.6. 88 1240 1328 0 1EP 1
12.6. 184 664 848 0 0 0 12.6. 59 630 689 0 0 0
23.6. 16 214 230 0 0 0 23.6. 58 491 549 0 1EP 1
30.6. 0 0 0 0 0 0 30.6. 44 403 447 0 0 0
7.7. 1 0 1 0 0 0 7.7. 5 169 174 0 0 0
15.7. 0 0 0 0 0 0 15.7. 12 38 50 0 0 0
22.7. 0 0 0 0 0 0 22.7. 0 0 0 0 0 0
30.10. 0 7 7 0 0 0 30.10. 0 0 0 0 0 0
11. Appendix
Table 55. Numbers of living and fungus-killed aphids of species Hyalopterus pruni (Geoffroy) recorded from 45 twigs of Prunus domestica Linné or 45
leaves of Phragmites australis (Cav.) Trin. at Dolná Malanta in 2002
Prunus domestica Linné Phragmites australis (Cav.) Trin.

2.4. 0 2 2 0 0 0 2.4. 0 0 0 0 0 0
9.4. 0 1 1 0 0 0 9.4. 0 0 0 0 0 0
16.4. 0 1 1 0 0 0 16.4. 0 0 0 0 0 0
2.5. 0 4 4 0 0 0 2.5. 0 0 0 0 0 0
9.5. 0 350 350 0 0 0 9.5. 0 0 0 0 0 0
14.5. 0 270 270 0 0 0 14.5. 0 0 0 0 0 0
21.5. 0 715 715 0 0 0 21.5. 0 0 0 0 0 0
28.5. 6 3498 3504 0 0 0 28.5. 0 0 0 0 0 0
4.6. 3 2450 2453 0 0 0 4.6. 105 791 904 0 0 0
12.6. 49 1117 1166 0 0 0 12.6. 283 466 749 0 0 0
19.6. 142 3100 3242 0 0 0 19.6. 106 1886 1992 1EP 0 1
26.6. 134 990 1124 0 0 0 26.6. 29 1031 1060 0 0 0
3.7. 1 3 4 0 0 0 3.7. 1 516 517 0 0 0
8.7. 0 0 0 0 0 0 8.7. 6 301 307 0 0 0
15.7. 0 0 0 0 0 0 15.7. 4 29 33 0 0 0
22.7. 0 0 0 0 0 0 22.7. 0 4 4 0 0 0
2.10. 0 0 0 0 0 0 2.10. 0 0 0 0 0 0
11. Appendix
Table 56. Numbers of living and fungus-killed aphids recorded from 45 plants of Pisum sativum Linné at Dolná Malanta in 2001 and 2002
Acyrthosiphon pisum (Harris) Acyrthosiphon pisum (Harris)

27.4. 0 0 0 0 0 0 9.4. 0 1 1 0 0 0
2.5. 0 5 5 0 0 0 16.4. 0 0 0 0 0 0
7.5. 1 13 14 0 0 0 25.4. 0 0 0 0 0 0
14.5. 0 17 17 0 0 0 2.5. 0 13 13 0 0 0
21.5. 1 38 39 0 0 0 9.5. 0 22 22 0 0 0
31.5. 1 70 71 0 0 0 14.5. 0 107 107 0 1PN 1
4.6. 0 5 5 0 0 0 21.5. 0 470 470 0 7PN 7
12.6. 1 24 25 0 1PN 1 28.5. 0 554 554 0 5PN 5
23.6. 0 31 31 0 1PN 1CO 2 4.6. 1 469 470 1PN 38PN 4CO 42
30.6. 0 19 19 1CO 1CO 2 12.6. 27 214 241 4PN 4CO 16PN 1CO 25
7.7. 1 138 139 0 0 0 19.6. 24 450 474 32PN 3CO 292PN 26CO 353
26.6. 2 390 392 4PN 2CO 99PN 14CO 119
11. Appendix
Table 57. Numbers of living and fungus-killed aphids of species Cavariella pastinacae (Linné) and Aphis fabae subg. mordwilkoi Börner et Janish recorded
from 45 twigs of Salix sp. or 45 leaves of Arctium lappa Linné, respectively, at Dolná Malanta in 2001
Cavariella pastinacae (Linné) Aphis fabae subg. mordwilkoi Börner et Janish

3.4. 0 13 13 0 0 0 7.5. 107 1985 2092 0 0 0
19.4. 0 255 255 0 0 0 14.5. 26 1265 1291 1PN 50NF 51
27.4. 0 322 322 0 0 0 21.5. 3 3340 3343 1NF 31NF 31
2.5. 14 321 335 0 0 0 31.5. 1 97 7 1NF 20NF 21
7.5. 18 1247 1265 0 1EP 1 4.6. 8 89 97 1NF 76NF 66
14.5. 47 2596 2643 0 0 0 12.6. 12 72 89 3NF 30NF 33
21.5. 189 1822 2011 0 0 0 23.6. 0 3 3 0 0 0
31.5. 100 1514 1614 2EP 0 2
4.6. 84 1106 1190 2EP 13EP 15
12.6. 7 149 156 0 1EP 1
23.6. 1 5 6 0 0 0
30.6. 0 0 0 0 0 0
11. Appendix
Table 58. Numbers of living and fungus-killed aphids recorded from 45 twigs of Padus avium Linné at Dolná Malanta in 2001 and 2002
Rhopalosiphum padi (Linné) Rhopalosiphum padi (Linné)

12.4. 0 328 328 0 0 0 13.3. 0 87 87 0 0 0
19.4. 0 666 666 0 0 0 21.3. 0 58 58 0 0 0
27.4. 0 528 528 0 1EP 1 28.3. 0 51 51 0 0 0
2.5. 10 175 185 0 0 0 2.4. 0 61 61 0 0 0
7.5. 1 71 72 0 0 0 9.4. 0 722 722 0 0 0
14.5. 3 19 22 0 5EP 5 16.4. 0 512 512 0 2EP 2
3.10. 283 215 498 3NF 1NF 3 25.4. 1 106 107 0 0 0
10.10. 665 1132 1797 18NF 10EP 2PN 30 2.5. 0 5 5 0 1EP 1
16.10. 633 1198 1831 49NF 32EP 1NF 81 20.9. 31 0 31 2NF 0 2
23.10. 126 714 840 30EP 15PN 10EP 5PN 60 27.9. 39 136 175 1CO 3NF 0 4
30.10. 44 281 325 5PN 10CO 35EP 2PN 3CO 10EP 65 2.10. 125 357 482 9NF 0 9
6.11. 15 37 81 10PN 6NF 2EP 4PN 5EP 27 17.10. 1151 7435 8586 191NF 64PN 1NF 255
14.11. 1 20 21 13PN 5NF 1NF 18 25.10. 1276 3205 4481 39NF 10ZA 29PN 13NF 14PN 115
1.11. 25 1140 1165 1PN 2PN 3
11. Appendix
Table 59. Numbers of living and fungus-killed aphids recorded from 45 twigs of Cerasus avium (Linné) Moench at Dolná Malanta in 2001 and 2002
Myzus cerasi (Fabricius) Myzus cerasi (Fabricius)

27.4. 0 45 45 0 0 0 21.3. 0 0 0 0 0 0
2.5. 0 161 161 0 0 0 2.4. 0 0 0 0 0 0
7.5. 0 319 319 0 0 0 9.4. 0 0 0 0 0 0
14.5. 2 2981 2983 0 0 0 16.4. 0 0 0 0 0 0
21.5. 9 1603 1612 0 1PN 1 2.5. 0 16 16 0 0 0
31.5. 1 222 223 0 2EP 2 9.5. 0 303 303 0 0 0
4.6. 1 101 102 0 0 0 14.5. 0 161 161 0 0 0
12.6. 0 0 0 0 0 0 21.5. 0 844 844 0 0 0
23.6. 0 50 50 0 0 0 28.5. 0 108 108 0 0 0
30.6. 0 0 0 0 0 0 4.6. 0 67 67 0 0 0
3.10. 1 0 1 0 0 0 12.6. 0 0 0 0 0 0
10.10. 1 5 6 0 0 0 17.10. 77 0 77 2NF 0 2
16.10. 4 7 11 0 0 0 25.10. 97 32 129 3ZA 4PN 1NF 0 8
30.10. 3 14 17 1EP 0 1 1.11. 28 33 61 3PN 0 3
6.11. 2 0 2 0 0 0
11. Appendix
Table 60. Numbers of living and fungus-killed aphids recorded from 45 plants of Triticum aestivum Linné at Dolná Malanta in 2001
Rhopalosiphum padi (Linné) Metopolophium dirhodum (Walker) Sitobion avenae (Fabricius)

19.4. 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 0 0 0
27.4. 0 0 0 0 0 0 0 2 2 0 0 0 0 1 1 0 0 0
2.5. 0 0 0 0 0 0 0 0 0 0 1PN 1 1 9 10 0 0 0
7.5. 0 0 0 0 0 0 0 0 0 0 0 0 10 36 46 1PN 0 1
14.5. 0 0 0 0 0 0 1 0 1 0 0 0 6 33 39 0 0 0
21.5. 1 35 36 0 0 0 0 11 11 0 0 0 0 26 26 0 1PN 1
31.5. 1 108 109 0 0 0 0 1 1 0 0 0 0 42 42 2PN 2PN 4
4.6. 0 3 3 0 0 0 0 0 0 0 0 0 0 2 2 0 1PN 1
12.6. 1 58 59 0 0 0 1 5 6 0 0 0 0 1 1 0 0 0
3.10. 0 0 0 2CO 0 2 0 0 0 0 0 0 0 0 0 0 0 0
30.10. 5 18 23 1PN 1NF 0 2 0 0 0 0 0 0 2 2 4 0 0 0
6.11. 4 12 16 2CO 0 2 0 0 0 0 0 0 1 3 4 0 0 0
11. Appendix
Table 61. Numbers of living and fungus-killed aphids recorded from 45 plants of Triticum aestivum Linné at Dolná Malanta in 2002
Rhopalosiphum Rhopalosiphum
padi (Linné) maidis (Fitch) Metopolophium dirhodum (Walker) Sitobion avenae (Fabricius)
2002 Living aphids Living aphids Living aphids Dead aphids Living aphids Dead aphids
9.5. 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 0 0 0
14.5. 0 0 0 0 0 0 0 5 5 0 0 0 0 7 7 0 0 0
21.5. 0 0 0 0 0 0 0 1 1 0 0 0 0 34 34 0 0 0
28.5. 0 0 0 0 0 0 2 114 116 0 0 0 4 109 113 0 0 0
4.6. 0 0 0 0 0 0 4 354 358 3PN 1PN 4 4 354 358 0 3PN 3
12.6. 0 2 2 0 2 2 6 357 363 0 10PN 10 6 357 363 0 3PN 1EP 4
19.6. 0 34 34 0 0 0 2 0 2 0 0 0 8 176 184 0 35PN 1EP 36
26.6. 0 0 0 0 0 0 0 0 0 0 1PN 1 0 1 1 1PN 8PN 9
11. Appendix
Table 62. Numbers of living and fungus-killed aphids recorded from 45 plants of Zea mays Linné at Dolná Malanta in 2001

31.5. 16 38 54 1NF 0 1 0 0 0 0 0 0 2 0 2 0 0 0
4.6. 18 70 88 3PN 0 3 2 9 11 0 0 0 2 16 18 1PN 0 1
12.6. 15 40 55 4PN 0 4 2 21 23 0 0 0 1 1 2 0 0 0
23.6. 5 40 45 0 0 0 2 8 10 0 0 0 0 3 3 0 0 0
30.6. 3 446 449 1PN 0 1 0 2 2 0 0 0 1 0 1 0 0 0
7.7. 21 1315 1336 1EP 2NF 3 0 1 1 0 0 0 1 0 1 0 0 0
15.7. 1 128 129 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
22.7. 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
17.8. 0 75 75 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
11.9. 0 73 73 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
19.9. 3 603 606 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
11. Appendix
Table 63. Numbers of living and fungus-killed aphids recorded from 45 plants of Zea mays Linné at Dolná Malanta in 2002

14.5. 1 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
21.5. 8 3 11 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
28.5. 1 5 6 0 0 0 36 477 513 0 0 0 0 0 0 0 0 0
4.6. 8 12 20 0 0 0 12 896 908 2PN 0 2 0 0 0 0 0 0
12.6. 19 141 160 1CO 0 1 390 1592 1982 193PN 1EP 12PN 206 0 0 0 0 0 0
19.6. 39 560 599 0 0 0 117 2522 2639 822PN 2NF 936PN 1787 1 0 1 0 0 0
26.6. 2 271 273 0 0 0 11 285 296 693PN 1308PN 2001 0 0 0 0 0 0
3.7. 3 53 56 0 0 0 1 15 15 101PN 414PN 515 0 0 0 0 0 0
8.7. 0 0 0 0 0 0 1 6 6 2PN 122PN 124 1 0 1 0 0 0
10.9. 0 1045 1045 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
20.9. 29 620 649 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
27.9. 1 230 231 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
11. Appendix
Table 64. Numbers of living and fungus-killed aphids recorded from 45 twigs of Acer campestre Linné at Dolná Malanta in 2001 and 2002
Periphyllus testudinaceus (Fernie) Periphyllus testudinaceus (Fernie)

3.4. 0 38 38 0 0 0 21.3. 0 5 5 0 0 0
12.4. 0 226 226 0 0 0 25.4. 1 43 44 0 0 0
19.4. 0 371 371 0 0 0 2.5. 1 42 43 0 0 0
27.4. 0 89 89 0 0 0 9.5. 1 11 12 0 0 0
2.5. 31 276 307 0 0 0 14.5. 2 0 2 0 0 0
7.5. 10 255 265 0 0 0 21.5. 0 2 2 0 0 0
14.5. 31 146 177 0 0 0 27.9. 4 124 128 0 0 0
21.5. 14 25 39 0 0 0 2.10. 6 145 151 0 0 0
31.5. 5 20 25 0 0 0 17.10. 6 221 227 0 0 0
16.10. 14 158 172 1PN 0 1 25.10. 4 67 71 1NF 0 1
23.10. 1 56 57 0 0 0 21.3. 0 5 5 0 0 0
30.10. 6 57 63 0 0 0 25.4. 1 43 44 0 0 0
6.11. 4 5 9 0 0 0 2.5. 1 42 43 0 0 0
11. Appendix
Table 65. Numbers of living and fungus-killed aphids of species Aphis fabae Scopoli recorded from 45 twigs (Euonymus europaea Linné) or plants (Faba
vulgaris Moench., Beta vulgaris Linné) at Dolná Malanta in 2001
Euonymus europaea Linné Faba vulgaris Moench. Beta vulgaris Linné

30.3. 0 567 567 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
12.4. 0 371 371 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
19.4. 0 1116 1116 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
27.4. 0 1254 1254 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
2.5. 37 1299 1336 1NF 0 1 6 0 6 1NF 0 1 0 0 0 0 0 0
7.5. 70 3422 3492 0 0 0 11 46 57 0 0 0 0 0 0 0 0 0
14.5. 124 1189 1313 0 0 0 5 199 204 0 0 0 0 0 0 0 0 0
21.5. 6 90 96 0 0 0 3 1781 1784 1NF 1NF 2 0 0 0 0 0 0
31.5. 0 0 0 0 0 0 1 15019 15019 1NF 10 NF 10 22 13 35 0 0 0
4.6. 0 0 0 0 0 0 174 9772 9946 1NF 113 NF 114 36 68 104 6 NF 0 6
12.6. 0 0 0 0 0 0 151 2380 2531 1NF 209 NF 209 14 1 15 1NF 2CO 0 3
23.6. 0 0 0 0 0 0 5 0 5 0 0 0
3.10. 5 60 65 0 0 0
10.10. 8 101 109 2EP 0 2
16.10. 43 488 531 1NF 0 1
23.10. 20 371 391 5EP 0 5
30.10. 35 545 580 8EP 0 8
6.11. 12 432 444 1EP 2EP 3
14.11. 7 161 168 7EP 0 7
11. Appendix
Table 66. Numbers of living and fungus-killed aphids of species Aphis fabae Scopoli recorded from 45 twigs (Euonymus europaea Linné) or plants (Faba
vulgaris Moench., Beta vulgaris Linné) at Dolná Malanta in 2002
Euonymus europaea Linné Faba vulgaris Moench. Beta vulgaris Linné

21.3. 0 5 5 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
28.3. 0 5 5 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
2.4. 0 27 27 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
9.4. 0 20 20 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
16.4. 0 142 142 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
25.4. 1 265 266 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
2.5. 2 477 479 0 0 0 3 22 25 0 0 0 0 0 0 0 0 0
9.5. 0 55 55 1EP 0 1 0 17 17 0 0 0 0 0 0 0 0 0
14.5. 0 0 0 0 0 0 2 86 88 0 0 0 0 0 0 0 0 0
21.5. 0 0 0 0 0 0 2 930 932 0 0 0 4 0 4 0 0 0
28.5. 0 0 0 0 0 0 15 4832 4847 0 0 0 6 60 66 0 0 0
4.6. 0 0 0 0 0 0 185 19748 19933 0 2NF 1PN 3 15 344 359 1NF 3NF 3
12.6. 0 0 0 0 0 0 686 15076 15762 0 3 3 1
NF PN CO 7 69 814 883 8 NF 22 NF 30
19.6. 0 0 0 0 0 0 159 987 1146 354NF 4PN 1178NF 7RS 2PN 1545
26.6. 0 0 0 0 0 0 5 125 125 92NF 1466NF 76RS 12PN 1646
3.7. 0 0 0 0 0 0 0 0 0 341RS10PN 351
10.9. 1 32 33 0 0 0
20.9. 22 52 74 0 0 0
27.9. 19 342 361 1NF 0 1 RS - aphids filled with resting spores of N. fresenii
2.10. 14 292 306 1NF 0 1
17.10. 16 290 306 0 0 0
25.10. 33 584 617 0 0 0
1.11. 3 13 16 0 0 0
11. Appendix
Table 67. Numbers of living and fungus-killed aphids recorded from 45 plants of Urtica dioica Linné at Dolná Malanta in 1998
Microlophium carnosum (Bukton) Dead aphids

1998 Living aphids Pandora neoaphidis Neozygites spp.
Date Alate Apteral ∑ Alate Apteral ∑ Alate Apteral RS ∑
24.5. 0 15 15 0 0 0 0 0 0 0
31.5. 0 50 50 0 8 8 0 0 0 0
3.6. 0 161 161 0 30 30 0 0 0 0
10.6. 33 953 986 6 290 296 1 45 0 46
15.6. 80 2149 2229 1 614 615 0 425 0 425
17.6. 38 1701 1739 29 695 724 16 483 0 499
19.6. 12 1333 1345 7 185 192 41 656 19 716
22.6. 14 2568 2582 9 149 158 18 574 3 595
25.6. 5 696 701 10 241 251 15 560 14 589
29.6. 15 1843 1858 7 297 304 11 343 5 359
1.7. 5 646 651 4 370 374 3 161 6 170
3.7. 1 131 132 5 147 152 0 105 3 108
8.7. 0 310 310 3 36 39 5 156 3 164
10.7. 0 484 484 5 53 58 7 256 4 267
13.7. 0 177 177 4 25 29 6 125 2 133
15.7. 0 176 176 4 14 18 9 71 0 80
20.7. 0 89 89 0 0 0 0 68 1 69
11. Appendix

7.5. 0 9 9 0 0 0 0 0 0 0
10.5. 0 2 2 0 0 0 0 0 0 0
17.5. 2 9 11 0 0 0 0 0 0 0
21.5. 49 171 220 0 0 0 0 0 0 0
24.5. 13 83 96 0 0 0 0 0 0 0
28.5. 18 109 127 0 0 0 1 0 0 1
31.5. 62 98 160 1 0 1 0 0 0 0
2.6. 80 309 389 2 0 2 0 0 0 0
4.6. 40 539 579 0 0 0 1 2 0 3
7.6. 41 1142 1183 2 0 2 5 0 0 5
9.6. 75 717 792 3 1 4 1 0 0 1
11.6. 50 1078 1128 3 2 5 3 4 0 7
16.6. 19 3182 3201 0 17 17 13 49 0 62
21.6. 0 1182 1182 4 146 150 6 26 0 32
24.6. 0 553 553 1 98 99 2 18 0 20
28.6. 0 116 116 4 193 197 3 36 3 42
30.6. 1 37 38 0 247 247 0 52 2 54
2.7. 0 11 11 1 118 119 2 29 1 32
11. Appendix

3.4. 122 0 122 10 0 10 0 0 0 0
12.4. 53 0 53 13 0 13 0 0 0 0
19.4. 59 0 59 35 0 35 0 0 0 0
27.4. 108 0 108 15 0 15 0 0 0 0
2.5. 84 0 84 72 0 72 1 0 0 1
7.5. 215 1 216 20 0 20 0 0 0 0
14.5. 106 5 111 23 0 23 0 0 0 0
21.5. 73 2 75 41 0 41 7 0 0 7
31.5. 664 0 664 23 1 24 0 2 0 2
4.6. 532 106 638 55+1CO 0 56 0 0 0 0
12.6. 231 1 232 138 2 140 27 0 0 27
23.6. 26 1 27 45+1EP 0 46 0 0 0 0
30.6. 2 0 2 8 0 8 0 0 0 0
7.7. 0 1 1 0 0 0 0 0 0 0
15.7. 0 0 0 0 0 0 0 0 0 0
11. Appendix

2.4. 0 0 0 0 0 0 0 0 0 0
9.4. 0 0 0 0 0 0 0 0 0 0
16.4. 5 0 5 0 0 0 0 0 0 0
25.4. 35 0 35 0 0 0 0 0 0 0
2.5. 12 0 12 0 0 0 0 0 0 0
9.5. 240 0 240 1 0 1 0 0 0 0
14.5. 301 0 301 49+19CO 0 68 0 0 0 0
21.5. 96 1 97 34+5CO 0 39 0 0 0 0
28.5. 117 2 119 10+8CO 0 18 0 0 0 0
4.6. 134 1 135 27+4CO 0 31 2 0 0 2
12.6. 67 1 68 4 0 4 0 0 0 0
19.6. 17 0 17 19+1CO 1 21 0 0 0 0
26.6. 0 0 0 2 0 2 0 0 0 0
11. Appendix
Table 71. Numbers of living and fungus-killed aphids recorded from 45 twigs of Cornus sanguinea Linné at Dolná Malanta in 2001 and 2002
Anoecia corni (Fabricius) Anoecia corni (Fabricius)

19.4. 0 210 210 0 0 0 21.3. 0 10 10 0 0 0
27.4. 0 147 147 0 0 0 2.4. 0 51 51 0 0 0
2.5. 0 515 515 0 0 0 9.4. 0 9 9 0 0 0
7.5. 62 787 849 0 0 0 16.4. 0 4 4 0 0 0
14.5. 0 2 2 0 0 0 25.4. 0 9 9 0 0 0
10.10. 739 1325 2064 0 0 0 2.5. 0 14 14 0 0 0
16.10. 699 1153 1852 0 0 0 9.5. 0 13 13 0 0 0
30.10. 648 943 1591 10ZA 0 10 14.5. 0 2 2 0 0 0
6.11. 117 314 431 55ZA 0 55 21.5. 0 0 0 0 0 0
14.11. 0 125 125 13ZA 0 13 1.9. 47 360 407 0 0 0
10.9. 10 176 186 0 0 0
20.9. 291 799 1090 2ZA 0 2
27.9. 121 600 721 8ZA 0 8
2.10. 351 1354 1705 1ZA 0 1
17.10. 102 1095 1197 6ZA 0 6
25.10. 576 1095 1671 3ZA 0 3
1.11. 21 135 153 3ZA 0 3
11. Appendix
Table 72. Numbers of living and fungus-killed aphids recorded from 45 plants of Hordeum vulgare Linné at Dolná Malanta in 2001
Diuraphis noxia (Mordvilko)

2001 Living aphids Dead aphids
Date Alate Apteral ∑ Alate Apteral ∑
8.6. 1 193 194 0 7PN 7
12.6. 1 292 293 0 1PN 1
23.6. 2 440 442 0 1EP 1PN 2
30.6. 10 1860 1870 0 7EP 7
7.7. 16 3775 3791 1PN 4PN 5
15.7. 26 727 753 0 102PN 102
11. Appendix
Table 73. Numbers of living and fungus-killed aphids recorded from 45 plants of Hordeum vulgare Linné at Dolná Malanta in 2002
Rhopalosiphum padi Metopolophium Sitobion avenae Rhopalosiphum maidis Diuraphis noxia

(Linné) dirhodum (Walker) (Fabricius) (Fitch) (Mordvilko)
Living Dead Living Dead Living Dead Living Dead Living Dead
2002 aphids aphids aphids aphids aphids aphids aphids aphids aphids aphids
Date Alate Apteral Alate Apteral Alate Apteral Alate Apteral Alate Apteral Alate Apteral Alate Apteral Alate Apteral Alate Apteral Alate Apteral
21.5. 0 0 0 0 0 12 0 0 0 1 0 0 0 0 0 0 0 0 0 0
28.5. 0 1 0 0 0 100 0 0 0 3 0 0 0 76 0 0 0 0 0 0
4.6. 0 0 0 0 6 243 1PN 3PN 0 83 0 0 2 76 0 0 0 0 0 0
12.6. 3 33 0 0 19 658 2PN 17PN 0 22 1PN 0 0 0 0 0 0 0 0 0
307PN
7PN 15PN 7PN
19.6. 5 223 0 14 328 1CO 4 47 1PN 13PN 17 194 1PN 0 2 0 0
10EP 1CO 1CO
1NF
26.6. 0 207 0 3PN 0 0 1PN 79PN 0 0 0 1PN 0 30 0 0 1 64 0 1PN
3.7. 0 19 0 0 0 0 0 0 0 0 0 0 0 55 0 0 2 111 1PN 11PN
8.7. 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 35 0 0

Fungi of The Order Entomophthorales Infecting Aphids in Slovakia

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Fungi of The Order Entomophthorales Infecting Aphids in Slovakia

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Fungi of the order Entomophthorales

infecting aphids in Slovakia

Faculty of Agrobiology and Food Department of Plant protection

Dean of the Faculty: Head of the Department:

prof. Ing. Magdaléna Lacko- prof. Ing. Ľudovít Cagáň, CSc.

Fungi of the order Entomophthorales infecting aphids in

4.3.5.3 Exposing of tested aphids to various quantities of inoculum………………………. 69

4.3.5.3Expozícia vošiek rôznym koncentráciám inokula………................………………. 69

2. AIMS OF THE WORK

 to collect information about entomophthoralean fungi associated with aphid populations in

 to carried out a survey of aphid colonies in natural ecosystems and agroecosystems to

 to compare the pathogen diversity in Slovakia with that in the world

 to obtain in vitro isolates of some aphid-pathogenic fungal species

 to examine some phenotypic features of P. neoaphidis isolates, including a primary

 to evaluate an occurrence of entomophthoralean fungi in selected aphid populations on

 to evaluate a possible role of non-crop vegetation as a reservoir of aphid-pathogenic

3.1 FUNGAL DISEASES OF ARTHROPODS

3.2 ENTOMOPHTHORALEAN INFECTION OF ARTHROPODS

3.3 CURRENT STATUS IN THE TAXONOMY OF ENTOMOPHTHORALES

Figure 1. Systematic position of Entomophthorales within the Kingdom of Fungi

Chytridiomycota Zygomycota Ascomycota Basidiomycota

CLASS Trichomycetes Zygomycetes

ORDER Mucorales Entomophthorales

Figure 2. Different classifications of arthropod-pathogenic fungi in the order Entomophthorales

Triplosporium  Neozygites  Neozygitaceae Neozygitaceae

3.4 APHIDOPHAGOUS ENTOMOPHTHORALES

Family: Entomophthoraceae Nowakowski, Bot. Ztg. 34 (14): 216-222, 1877

1. Pandora neoaphidis (Remaudière et Hennebert) Humber, Mycotaxon 34: 452, 1989

2. Pandora delphacis (Hori) Humber, Mycotaxon 34: 452, 1989

3. Pandora nouryi (Remaudière et Hennebert) Humber, Mycotaxon 34: 453, 1989

Zoophthora subgenus Neopandora nouryi (Remaudière et Hennebert) Ben-Ze’ev et Kenneth sensu

4. Pandora kondoiensis (Milner) Humber, Mycotaxon 34: 453, 1989

Genus: Erynia (Nowakowski ex Batko) Remaudière et Hennebert emend. Humber,

5. Erynia erinacea (Ben-Ze’ev et Kenneth) Remaudière et Hennebert, Mycotaxon 11:

6. Erynia conica (Nowakowski) Remaudière et Hennebert, Mycotaxon 11: 302, 1980

7. Zoophthora aphidis (Hoffman in Fresenius) Remaudière et Hennebert, Mycotaxon 11:

8. Zoophthora phalloides Batko, Acta Mycol. 2: 7-13, 1966a

survey of entomopathogens in aphid populations infesting natural vegetation in France

The species Z. radicans was originally described as a pathogen of caterpillar, Pieris

10. Zoophthora canadensis (MacLeod, Tyrrell et Soper) Remaudière et Hennebert,

12. Zoophthora orientalis Ben-Ze’ev et Kenneth, Phytoparasitica 9 (1): 33-42, 1981

13. Zoophthora anhuiensis (Li) Humber, Mycotaxon 34: 453, 1989

Genus: Entomophaga Batko emend. Humber, Mycotaxon 34: 447-448, 1989

Genus: Batkoa Humber, Mycotaxon 34: 446-447, 1989

17. Batkoa apiculata (Thaxter) Humber, Mycotaxon 34: 446, 1989

Macrosiphoniella mutellinae Börner, Sitobion avenae (Fresenius) and Rhopalosiphum padi

18. Batkoa major (Thaxter) Humber, Mycotaxon 34: 446, 1989

This fungus, found on naturally infected adults of Ptilodactyla serricollis (Say)

Family: Neozygitaceae Ben-Ze’ev et Kenneth in Ben-Ze’ev et al., Mycotaxon 28: 313-326,

reference to this fungus /GUSTAFSSON 1969/ referred to it as a new species Entomophthora

20. Neozygites microlophii Keller, Sydowia 43: 82, 1991

The pathogen was discovered in Israel on aphid Pterochloroides persicae (Cholodk.) in

In Switzerland the species was observed in colonies of aphid Tuberolachnus salignus

24. Neozygites cinarae Keller, Sydowia 49 (2): 137-138, 1997

Family: Basidiobolaceae Claussen in Engler et Gilg, Syll. Pflanzenfam.: 45, 1924

25. Basidiobolus ranarum Eidam, Beitr. Biol. Pflanz. 4: 194-239, 1886

Family: Ancylistaceae v. Thieghem, Traité de Botanique: 1007, 1884

insects, in which death resulted from infection by a parasitic Entomophthorales or from

C. thromboides is best known as an aphid pathogen, but it was first described as a

29. Conidiobolus osmodes Drechsler, Am. J. Bot. 41: 571, 1954

This species was originally described as a saprophyte on plant detritus by DRECHSLER

recorded as an occasional pathogen of aphids in France, it was recorded as a very common

Form-genus: Tarichium Cohn, Beitr. Biol. Pflanz. 1: 58-86, 1870